Perspectives of immobilized-metal affinity chromatography

Ž .J. Biochem. Biophys. Methods 49 2001 335–360www.elsevier.comrlocaterjbbm

Review

Perspectives of immobilized-metalaffinity chromatography

Vladka Gaberc-Porekara,), Viktor Menartb

a National Institute of Chemistry, HajdrihoÕa 19, SI-1000 Ljubljana, SloÕeniab Lek d.d., R&D, Biotechnology Department, CeloÕska 135, SI-1000, Ljubljana, SloÕeniaˇ

Abstract

Ž .Immobilized Metal-Affinity Chromatography IMAC represents a relatively new separationtechnique that is primarily appropriate for the purification of proteins with natural surface-exposedhistidine residues and for recombinant proteins with engineered histidine tags or histidine clusters.Because the method has gained broad popularity in recent years, the main recent developments inthe field of new sorbents, techniques and possible applications are discussed in this article.Advantages of the method and new prospects are described as well as the problems and concernsthat appear when the method is to be used for production of pharmaceutical-grade proteins.q2001 Elsevier Science B.V. All rights reserved.

Keywords: IMAC; Immobilized-metal affinity chromatography; Recombinant proteins; Histidine tags; Phar-maceutical proteins

1. Introduction

Ž .Immobilized-Metal Affinity Chromatography IMAC is a separation technique thatuses covalently bound chelating compounds on solid chromatographic supports to entrapmetal ions, which serve as affinity ligands for various proteins, making use of coordina-tive binding of some amino acid residues exposed on the surface. As with other forms ofaffinity chromatography, IMAC is used in cases where rapid purification and substantialpurity of the product are necessary, although compared to other affinity separationtechnologies it cannot be classified as highly specific, but only moderately so. On the

) Corresponding author. Tel.:q386-1-476-0200; fax:q386-1-425-9244.Ž .E-mail address: [email protected] V. Gaberc-Porekar .

0165-022Xr01r$ - see front matterq2001 Elsevier Science B.V. All rights reserved.Ž .PII: S0165-022X 01 00207-X

( )V. Gaberc-Porekar, V. MenartrJ. Biochem. Biophys. Methods 49 2001 335–360336

other hand, IMAC holds a number of advantages over biospecific affinity chromato-graphic techniques, which have a similar order of affinity constants and exploit affinitiesbetween enzymes and their cofactors or inhibitors, receptors and their ligands orbetween antigens and antibodies. The benefits of IMAC—ligand stability, high protein

w xloading, mild elution conditions, simple regeneration and low cost 1 —are decisivewhen developing large-scale purification procedures for industrial applications.

w xEverson and Parker 2 were the first who adapted immobilization of chelatingcompounds to the separation of metalloproteins. The method became popular through

w x w xthe research work of Porath 3–7 and Sulkowski 8–12 who laid the foundations of thetechnique that is widely used today and applicable for a variety of purposes, includinganalytical and preparative purification of proteins, as well as being a valuable tool forstudying surface accessibility of certain amino acid residues. Initially, IMAC techniqueswere used for separating proteins and peptides with naturally present, exposed histidineresidues, which are primarily responsible for binding to immobilized metal ions.

w xHowever, the work of Hochuli et al. 13,14 pioneered the efficient purification ofrecombinant proteins with engineered histidine affinity handles attached to the N- or

Ž . Ž .C-terminus, especially in combination with the Ni II –nitrilotriacetic acid Ni–NTAmatrix, which selectively binds adjacent histidines. Since numerous neighboring histi-dine residues are uncommon among naturally occurring proteins, such oligo-histidineaffinity handles form the basis for high selectivity and efficiency, often providing aone-step isolation of proteins at over 90% purity.

Another distinct advantage of this kind of IMAC over biospecific affinity techniquesis its applicability under denaturing conditions. This is often necessary when recombi-nant proteins are highly expressed inE. coli in the form of inclusion bodies. Whenappropriate cleavage sites are engineered between the affinity tags and proteins, with thepurpose of enabling effective and precise tag removal after the main isolation step,IMAC seems to be an ideal solution for many applications. However, for the productionof therapeutic proteins in substantial quantities, multiple operational cycles with highreproducibility are required as well as the minimal leaching of metal ion, exactterminals, and defined minimal values of host cell proteins, DNA, endotoxins, viruses,etc. To this end, the principles of the method have been studied intensively, andnumerous modifications have been made for specific purposes. Because theoretical andpractical issues of IMAC have already been widely reviewed by several authorsw x1,7–9,15,16 , this short report will focus on novel uses and problems that have surfacedin recent years.

2. Mechanism, ligands, ions, and techniques

In IMAC the adsorption of proteins is based on the coordination between animmobilized metal ion and electron donor groups from the protein surface. Fig. 1illustrates protein binding to a metal-chelated affinity support. Most commonly used are

Ž . Ž . Ž . Ž . Ž .the transition-metal ions Cu II , Ni II , Zn II , Co II , Fe III , which are electron-pairŽ .acceptors and can be considered as Lewis acids. Electron-donor atoms N, S, O present

( )V. Gaberc-Porekar, V. MenartrJ. Biochem. Biophys. Methods 49 2001 335–360 337

Fig. 1. Schematic illustration of the protein binding to a metal-chelated affinity support. Strong binding of aprotein onto the IMAC matrix is achieved predominately by multi-point attachment of native or engineered

Ž . Ž .surface histidines a , or by histidine tag b added to the N- or C-terminus of the protein. There are manypossibilities for the construction of efficient His tags considering the number of histidines, their location andmicroenvironment. However, in practice some simple solutions consisting of consecutive histidine residues,e.g., His6 or His10, prevail.

in the chelating compounds that are attached to the chromatographic support are capableof coordinating metal ions and forming metal chelates, which can be bidentate,tridentate, etc., depending on the number of occupied coordination bonds. The remainingmetal coordination sites are normally occupied by water molecules and can be ex-changed with suitable electron-donor groups from the protein. In addition to the aminoterminus, some amino acids are especially suitable for binding due to electron donoratoms in their side chains. Although many residues, such as Glu, Asp, Tyr, Cys, His,Arg, Lys and Met, can participate in binding, the actual protein retention in IMAC isbased primarily on the availability of histidyl residues. Free cysteines that could alsocontribute to binding to chelated metal ions are rarely available in the appropriate,reduced state. However, aromatic side chains of Trp, Phe and Tyr appear to contribute to

w xretention, if they are in the vicinity of accessible histidine residues 1,9 .Adsorption of a protein to the IMAC support is performed at a pH at which imidazole

nitrogens in histidyl residues are in the nonprotonated form, normally in neutral orŽslightly basic medium. Usually relatively high-ionic-strength buffers containing 0.1 to

.1.0 M NaCl are used to reduce nonspecific electrostatic interactions, while the bufferitself should not coordinatively bind to the chelated metal ion. Elution of the targetprotein is achieved by protonation, ligand exchange or extraction of the metal ion by astronger chelator, like EDTA. Elution buffers with lower pH or lowering pH gradients


are widely used for elution of the target protein. However, for proteins sensitive to lowpH, ligand exchange, e.g., with imidazole, at nearly neutral pH is more favorable. In thiscase, the IMAC columns must be saturated and equilibrated with imidazole prior tochromatographic separation to avoid the pH drop caused by the imidazole proton pump

w xeffect 10,11 . Application of a strong chelating agent, such as EDTA, also results inelution of the bound proteins, although the binding properties are also destroyed and thecolumn must be recharged with metal ions prior to the next separation.

Selectivity in protein separation can be effected through various approaches: bychoice of the metal ligand, through variation of the structure of the chelating compound,by variation of the spacer arms, ligand density, concentration of salts and competingagents, etc. For example, in the case of human growth hormone, reduction of ligand

Ž .IDA–Cu II density on chelating sorbent resulted in higher protein purity and increasedw xyield 17 . The apparent affinity of a protein for a metal chelate depends strongly on the

Ž .metal ion involved in coordination. In the case of the iminodiacetic acid IDA chelator,the affinities of many retained proteins and their respective retention times are in the

Ž . Ž . Ž . Ž .following order: Cu II )Ni II )Zn II GCo II . In contrast to these currently mostcommonly used metal ions, which have a preference for extra-nitrogen-containing amino

Ž . Ž . Ž . Ž .acids, hard Lewis metal ions, such as Al III , Ca II , Fe III , Yb III , prefer oxygen-richw xgroups of aspartic and glutamic acid or phosphate groups 5,18 , and this provides an

opportunity to engineer new affinity handles, based on glutamic- or aspartic-acid-richaffinity tails.

IDA is by far the most widely used chelating compound. It is commercially availablefrom many producers, although in the past several years, other chelators have also been

Ž .tried for immobilization to support particles Table 1 . In general, tetradentate ligands,Ž .such as NTA and TALON trade name for carboxymethylated aspartic acid: CM-Asp ,

have higher affinities for metal ions than the tridentate chelator IDA, but they exhibitlower protein binding due to the loss of one coordination site. This is even morepronounced in a pentadentate TED chelating ligand, where in an octahedral arrangementaround a divalent metal ion only one coordination site is left for protein binding.Putative structures of metal ion complexes and most popular chelators are shown in Fig.2.

Classical stationary phases are based on soft-gel matrices, such as agarose orcross-linked dextran. While polysaccharides are biologically compatible and easilyactivated, they exhibit low mechanical strength and a large pressure drop, which limitstheir use in large-scale industry. On the other hand, inorganic adsorbents, like silica,have excellent mechanical properties but exhibit irreversible nonspecific adsorption ofproteins. Combination of polysaccharide on inorganic beads has resulted in several new

Ž .supports that are also applicable to the expanded-bed adsorption EBA technique, whichenables the recovery of proteins directly from unclarified cell suspensions and ho-

Ž .mogenates. In Streamline Chelating Amersham Pharmacia Biotech the defined particledistribution is achieved by combining macroporous, cross-linked agarose with a crys-

Žtalline quartz core and dextran coating, while UpFront EBA adsorbents UpFront.Chromatography, Copenhagen are based on highly cross-linked agarose beads with a

central core of high-density glass. Silica-based or synthetic polymer-based particles,Ž . Ž .such as Ni–NTA silica Qiagen , Prosep-Chelating Bioprocessing or TSKgel Chelate

()

V.G

aberc-Porekar,V

.Menartr

J.Biochem

.Biophys.M

ethods49

2001335

–360

339

Table 1Some chelating compounds in use for immobilization in IMAC

Chelating compound Coordination Metal ions Reference Commercial source

Ž . w xAminohydroxamic acid bidentate Fe III 5Ž . w xSalicylaldehyde bidentate Cu II 113

Ž . Ž . Ž . Ž . w x8-Hydroxy-quinoline 8-HQ bidentate Al III , Fe III , Yb III 114Ž . Ž . Ž . w xIminodiacetic acid IDA tridentate Cu II , Zn II , 3,4 Amersham Pharmacia Biotech, Uppsala; Pierce,

Ž . Ž .Ni II , Co II Rockford, IL; Sigma, St. Louis, MO; BoehringerMannheim; Bioprocessing, Princeton, NJ; TosoHaas,Montgomeryville, PA; Merck, Darmstadt

Ž . Ž . Ž . w xDipicolylamine DPA tridentate Zn II , Ni II 7Ž . Ž . Ž . w xOrtho-phosphoserine OPS tridentate Fe III , Al III , 115

Ž . Ž .Ca II , Yb IIIŽ . Ž . w xN- 2-pyridylmethyl tridentate Cu II 35

aminoacetateŽ . w x2,6-Diaminomethylpyridine tridentate Cu II 36

Ž . Ž . w xNitrilotriacetic acid NTA tetradentate Ni II 13,14 Qiagen, Chatsworth, CAŽ . Ž . w xCarboxymethylated aspartic tetradentate Ca II , Co II 7,38,58,116 Clontech, Palo Alto, CA

Ž .acid CM-AspX Ž . Ž . Ž . w xN,N,N -tris carboxymethyl pentadentate Cu II , Zn II 7

Ž .ethylenediamine TED


Fig. 2. Putative structures of some representative chelators in complex with usually used metal ions:Ž . Ž . Ž . Ž . Ž . Ž . Ž . Ž . Ž .IDA–Me II , NTA–Ni II , CM–Asp–Co II , TED–Me II . Me II stands for Cu II , Ni II , Zn II or Co II .

Spacers to the solid support are not specified, but may vary in length and chemical structure, which alsoaffects chromatographic behavior. Water molecules can be replaced by other ligands, usually histidinesexposed on the protein surface. This represents the major binding interaction of the protein towards the IMACmatrix, provided that unspecific, residual interactions, e.g., ionic or hydrophobic, are minimized by selection

Ž w xof appropriate matrix material and buffer composition. This figure was adapted according to Ref. 13 and.templates of Dr. L. Jacob .

Ž .TosoHaas can withstand high pressures in HPLC systems and can also be used forexpanded-bed technology, which is especially appropriate for large-scale separations dueto the reduced number of steps and consequently decreased process time. To increase theefficiency of protein binding, tentacle gels were developed, e.g., Fractogel EMD ChelateŽ .Merck . Coupling of a chelator molecule, such as IDA, to the linear polymer chainsŽ .tentacles , results in higher functional-group density and better steric accessibility dueto increased flexibility of polymer chains as compared to the conventional spacertechnology. As a result, higher protein capacity and stronger binding of the protein isachieved.

Membranes consisting of a hydrophylic copolymer and carrying metal chelatingŽ .groups, the so-called Immobilized-Metal Affinity Membrane Adsorbers IMA-MA ,

represent an interesting alternative to conventional chromatography, especially in termsw xof speed and simple scale-up 19 . Some years ago these membranes were commercially


Ž .available from Sartorius Goettingen but they are not marketed any more. IDA was alsobound to the surface of the Bioran-M glass hollow fiber microfiltration membranesŽ .Schott Glass, Mainz , which exhibited higher metal loading capacity than conventional

w xIMAC matrices but lower protein binding 20 . Recovery of serum proteins using porousŽ . w xIDA–Cu II cellulose affinity membranes was also demonstrated 21 . The role of a

spacer element between the polymeric backbone and chelating group IDA was studiedŽ .on microporous sheets obtained from Arbor Tech Ann Arbor, MI . Membranes with no

Ž .spacer and with 14-atom spacing element 1,4-butanediol diglycidyl ether , bearing IDA,Ž .were prepared and charged with Cu II . Equilibrium adsorption of lysozyme was similar

for both membrane types. However, dynamic adsorption was much higher when thew xspacer was included 22 .

Ž .Recently, an interesting application of IDA–Cu II polysulfone hollow-fibre mem-w xbrane for fractionation of the commercial pectic enzyme has been reported 23 .

Pectinlyase, useful for large-scale fruit-juice clarification, passed through the membrane,while pectinesterase, responsible for undesirable methanol production, was retained. Anarticle on the combined dye and metal chelate affinity membranes has also been

w x Žpublished 24 . Cibacron Blue F3GA was covalently bound to microporous poly 2-hy-. Ž .droxyethyl methacrylate membrane and charged with Fe III ions to study

adsorptionrdesorption behavior of various pure proteins. The same type of membraneswas used for immobilization of glucose oxidase and the potential for the construction of

w xglucose biosensors was demonstrated 25 . Cellulose membranes with imidazole as aŽ . w xligand for Cu II immobilization have also been described 26 . In general, affinity

membranes operate in convective mode, which can significantly reduce diffusionlimitations commonly encountered in column chromatography. As a result, higherthroughput and faster processing times are possible in membrane systems. Membranesare also capable of handling unclarified solutions and thus can be applied in the earlierstages of downstream processing. Therefore, wide applicability of IMA membranes forhigh-speed purification and analyses as well as for capture of trace amounts ofhistidine-bearing impurities, would be expected. However, to our knowledge, there areno commercially IMA membranes available.

Another example of novel IMAC stationary phases are various metalloprotopor-w xphyrins, covalently bound to silica supports 27 , which have demonstrated a fundamen-

tal advantage of stable metal binding over the normally used stationary phases withweakly anchored metal ions.

3. Purification of proteins

3.1. NatiÕe-surface histidines

Numerous natural proteins contain histidine residues in their amino acid sequence.However, histidines are mildly hydrophobic and only few of them are located on theprotein surface. For proteins with known 3D structure, data about the number and


arrangement of surface histidine residues can be obtained from protein data banks. Thiscan also serve as a basis for forecasting their behavior in IMAC. In the coming years,with the development of proteomics, the number of proteins with known primarystructure is bound to grow much faster than the number of 3D structures resolved, butstructure modeling, based on the known primary amino acid sequence, will also becomemore useful and more accurate. However, until now, no data from systematic searches ofthe protein data bases regarding surface histidines have been published.

For use in IMAC, protein-surface histidine residues must also be accessible to themetal ions and their bulky chelating compounds. However, the microenvironment of thebinding residue, cooperation between neighboring amino acid side groups, and local

Ž .conformations play important roles in protein retention Fig. 3 . In this way, IMAC canserve as a sensitive tool for revealing protein topography with respect to histidines and

w xtheir surroundings 6,8 . Depending on proximity and orientation of histidines and

Fig. 3. High resolving power of IMAC demonstrated by separation of TNF-a and its histidine analogs. AŽ . Žmixture of equal amounts 0.1 mg of purified TNF-a and its three histidine analogs His108, His107 and

. Ž . Ž .His107His108 was separated on IDA–Cu II Chelating Superose Pharmacia, Uppsala using an optimizedimidazole gradient. Histidines were introduced in the exposed loop region on the tip of trimeric TNF-a. Topviews of the trimeric TNF-a and its analogs are schematically depicted, showing different number and

Ž . Ž .arrangement of histidines. Most interestingly, analogs His108 G108H and His107 E107H have equalnumber of completely exposed histidines, however, due to different microenvironment the retention times of

w xthese analogs are very different, indicating the power of IMAC for surface topography studies 59 . Legend:Ž . Ž . Ž .asterisk ) Glu; open circle ` Gly; dot v His.


density of the chelating groups and metal ions, as well as on spatial accessibilitiesbetween the support particles and protein, multipoint binding of different histidines can

w xbe achieved 16 . In general, the protein shows the highest affinity for the metal surfacearrangement which best matches its own distribution of functional histidines. Adjacenthistidines can bind to the same or different chelating sites. Usually, one histidine is

Ž .enough for weak binding to IDA–Cu II , while more proximal histidines are needed forŽ . Ž .efficient binding to Zn II and Co II . Some interesting examples of using IMAC for

w xproteins with naturally exposed histidine residues include human serum proteins 3,4,28 ,w x w x w xinterferon 8 , lactoferrin and myoglobin 11 , tissue plasminogen activator 29 , antibod-

w x w xies 30,31 , and yeast alcohol dehydrogenase 32 . In general, a positive correlation isw xfound between the number of accessible histidines and the strength of binding 8 .

Separation of gamma-chymotrypsin, a common contaminant in commercial alpha-w xchymotrypsin, was achieved by IMAC, due to various number of surface histidines 33 ,

indicating possible industrial application. Interesting IMAC behavior is exhibited byw xnatural cytochromeC from different species, which differ in their histidine content 6 .

Similarly, evolutionary variants of the lysozymes show varied affinities for IMACw xmatrices due to differences in the surface topography of histidines 34 . On the other

hand, albumins contain up to 16 histidine residues in their structure but only onew xhigh-affinity binding site His3 at the N-terminus 8 . Recently, human serum proteins

w xhave been used for testing new IMAC affinity ligands 35,36 .

3.2. Histidine tags

Although the first demonstrations of IMAC were low-resolution group separations,the resolution has significantly improved with the use of genetically engineered affinitytags that can be attached to amino or carboxy terminals of the recombinant proteins. Thefirst histidine-rich fusions were made on the basis of the high affinity of certain naturalproteins containing histidine residues near the N-terminus. For instance, an octapeptidederived from angiotensine I was fused to the N-terminus of the TEM-b-lactamase andexpressed inE. coli in the form of inclusion bodies. One-step purification of therecombinant protein from the resolubilized inclusion body material was achieved on

Ž . w xIDA–Zn II 37 . Recently, a natural amino acid sequence, located on the N-terminus ofchicken lactate dehydrogenase, has been described which is responsible for efficient

Ž .binding to Co II -carboxymethylaspartate IMAC. The natural peptide contains sixw xhistidines, unevenly interleaved by other amino acid residues 38 . Its truncated version,

Ž . Ž .designated as histidine affinity tag HAT Table 2 , was fused to the N-terminals ofw xthree recombinant proteins to demonstrate its utility as a purification tag 39 . In the

past, numerous histidine tags were employed, from very short ones, e.g., HisTrp, utilizedw xfor isolation of sulfitolized proinsulin 40 to rather long extensions, containing up to

eight repeats of the peptide Ala-His-Gly-His-Arg-Pro, attached to various model proteinsw x41 . However, today by far the most widely used histidine tags consist of 6 consecutive

X w xhistidine residues. After the appearance of Hochuli s papers 13,14 , describing a newchelating matrix Ni–NTA and fusions with short peptides, containing 2–6 neighboringhistidines, these hexa-histidine tags have become very popular. Commercial expres-sion vectors, containing nucleotides coding for His6, His10, and some other fusions

()

V.G

aberc-Porekar,V

.Menartr

J.Biochem

.Biophys.M

ethods49

2001335

–360

344

Table 2Some commercially available expression systems encoding various histidine-rich affinity tags

Expression system Histidine tag Cleavage Immunodetection of the Commercial sourcehistidine tag

w Ž .QIAexpress systemsr H extensions at N- or C-terminus no cleavage at N- or C-terminus PentaPHise mAb; Qiagen6

E. coli RGSPHise mAb;TetraPHise mAb

Ž .pcDNA, pEF, etc. seriesr H extensions at N- or C-terminus enterokinase at N-terminus anti-HisG mAb; Invitrogen, Carlsbad, CA6Ž .mammalian cells anti-His C-term mAb

Ž . Ž .pMET and pPICZ seriesr H extensions at C-terminus no cleavage anti-His C-term mAb Invitrogen6

methylotrophic yeastsŽ . Ž .pYES seriesr H extensions at C-terminus no cleavage anti-His C-term mAb Invitrogen6

classical yeastwŽ .pTriEx vectorsr Protein- H no cleavage HisPTag mAb Novagen, Madison, WI8

E.coli, baculovirusand mammalian cells

wŽ .pET systemsr MG H SSGHIDDDDKxH-Protein enterokinase HisPTag mAb against Novagen10

E. coli N- or C-terminal His tagsŽ .MG H SSGHIEGRxH-Protein factor X10

Ž .MGSS H SSGLVPRGSxH-Protein thrombin6

Ž .Protein- H no cleavage6

pHAT vectorsr MKDHLIHDVHKEEHAHAHNKI- enterokinase HAT Polyclonal Ab ClontechE. coli DDDDKx -Protein–

Ž . Ž .TAGZyme kitr MK HQ Qx-Protein and various, dipeptidyl aminopeptidase I DPP I – UNYZYME Laboratories,6

E. coli other His tags optimized for this kit alone or a combination of DPP I, HørsholmŽ .glutamin cyclotransferase GCT

and pyroglutamyl aminopeptidaseŽ .PGAP


Ž .Table 2 , have been on the market for several years. However, His10 tags, even thoughw xefficient 42 , have never received as much attention as His6. There is a very large

w xnumber of papers on the use of His6 tag 13,14,43–47 . Recently, a versatile strategyusing His6–GFP, fused to the target protein, has been published, enabling simplefluorescence monitoring of the expression, and localization, as well as easy purification

w xof the fusion protein by IMAC 44,48 . The principle of polyhistidine tags is based onthe premise that multiplicity of histidines may increase binding. On the other hand, veryhigh affinity, which is an absolute requirement in some immobilized-metal-ion-based

Ž .nonchromatographic techniques single-stage processes, such as partitioning , is notw xalways advantageous in chromatographic multi-stage processes 1 . An ideal affinity tag

should enable effective but not too strong a binding, and allow elution of the desiredprotein under mild, nondestructive conditions. In the case of recombinantE. coli, manyhost proteins strongly adhere to the IMAC matrices, especially when charged with

Ž . Ž .Cu II or Ni II ions, and are eluted with the target proteins. Therefore, new approachesfor selecting improved histidine tags have focused on elution of the target protein in theAcontaminant-freeB window. Interestingly, selection of an optimum tag by a phage-dis-played library showed that tags with only two histidine residues possessed chromato-

w xgraphic characteristics superior to those of the most commonly used His6 tag 49,50 .Ž . Ž .Similarly, in many cases, IDA–Zn II may prove superior to either immobilized Cu II

Ž . w xor Ni II ions, as a result of its relatively low binding affinity for host cell proteins 51 .Oligomeric proteins, as for example trimeric TNF-a , pose additional difficulties whenone is searching for useful affinity tags, since interactions with the matrix are multipliedw x52 .

A different approach to achieving selective adsorption of engineered oligo-histidine-tagged proteins with minimal interference of host cell proteins involvesAtailor-madeBchelating supports with very short spacer arms and low surface density of chelating

w xgroups 53 .Histidine tags seem to be compatible with all expression systems used today. Thus,

His-tagged proteins can be successfully produced in procaryotic and eucaryotic organ-Ž . w xisms Table 2 , intracellularly or as secreted proteins 54 . The use of long histidine tags

in E. coli cells may reduce the accumulation level or induce the formation of inclusionŽ .bodies of otherwise soluble protein Menart, unpublished results . However, which

position is preferable for the addition of His tag, N- or C-terminus, depends on thenature and intended use of the protein, and must be determined experimentally. Additionof His tag to the N-terminus of the protein appears to be more universal, if judged fromthe huge number of cases reported. Most likely, N-terminal tagging is more frequentlyused because several efficient endoproteases are available for precise cleavage of the tagafter purification. Histidine tagging and IMAC have become a routine for easy first-timeisolation of newly expressed proteins. In most cases, the histidine tags neither affectprotein folding, nor interfere significantly with the biological functionality.

3.3. Designed histidine patches and motifs

In contrast to histidine tags, the possibility of engrafting new surface histidines foreasy purification depends very much on the intended use of the protein. In therapeutic


Ž .Fig. 4. Single-step IMAC isolation of TNF-a analog His107His108 E107H,G108H from theE. coliŽ . w x Ž . Ž .homogenate ammonium sulfate precipitate 59 . a Chromatographic separation on IDA–Cu II Chelating

Ž . Ž . Ž . ŽSuperose Pharmacia . b SDS-PAGE analysis silver stain ; St—Standard LMW, Bio-Rad, Hercules, CA,.and TNF–a ; S—Sample; P—protein composition of the last chromatographic peak containing more than

95% pure analog.


proteins there is a serious limitation of this approach because an authentic surface of theŽ .protein is usually required. Also 3D structure and active site s must be well character-

ized if one intends to design a protein with the desired affinity towards the chosenIMAC matrix. In many cases, a high enough affinity can be achieved when two or moresurface histidines lie approximately in a plane. Thus, a concerted attachment of allexposed histidines is possible. Flexible loops are among the most attractive regions forthe introduction of new histidines or for the replacement of existing amino acid residues.However, no universal rule exists and every protein and its 3D structure represent aspecial case. Therefore, we mention here just a few examples.

After recognizing that some high-affinity natural binding sites, such as His-X -His3Žtwo histidines separated by a turn ina-helical structure, as in myoglobin or human

. Ž .fibroblast interferon , are most probably responsible for binding to IDA–Zn II andŽ . w xIDA–Co II 8,12 , these sequences were engineered into cytochromeC and bovine

w xsomatotropin 1,55 . The mutant proteins actually demonstrated higher affinity. Simi-Ž .larly, the Zn II -binding site of human carbonic anhydrase, which includes three

histidine residues, was successfully engineered onto the surface of the retinol-bindingw xprotein 56 . In general, the sites for introducing histidine residues must be exposed and

separated structurally from the active site of the protein. Thus, their design is most easilyaccomplished when the biochemical properties and 3D structure are known. Anothersuccessful example of a newly introduced histidine cluster consists of mutants of

w xglutathione transferase 57 , constructed on the basis of the natural rat enzyme thatcontains two adjacent histidine residues forming a four-histidine cluster on the surface of

w xthe dimeric protein 58 . A similar effect was achieved by introducing one or twohistidine residues into the flexible-loop region of the trimeric molecule of TNF-a

w x52,59 , which resulted in planar surface clusters of three or six histidines and very goodŽ .chromatographic characteristics in IMAC matrices Fig. 3 and Fig. 4 . Although such

newly designed histidine clusters can be very effective for rapid purification and canalso be used for immobilization purposes, the engineered proteins are mutants whichdiffer to a greater or a lesser extent from the authentic structures with respect toimmunogenicity, biological activity, stability, etc. However, this approach could be veryuseful for many other groups of proteins not intended for human therapy, e.g., industrialenzymes, proteins for diagnostic purposes, and enzymes for research.

4. Large-scale purification of therapeutic proteins

Many reports on IMAC used for purifying pharmaceutically interesting proteins, suchas interferons, vaccines and antibodies, have been published but relatively few data existon actual large-scale purifications of pharmaceutical proteins. On the other hand, IMACoffers all possibilities for large-scale purification of many industrial enzymes as well asproteins for research in genetics, molecular biology, and biochemistry.

Recently, some interesting reports on IMAC techniques used for purifying vaccineshave appeared. For example, an efficient purification procedure for malaria vaccine

w xcandidates, expressed as His6-tagged proteins inE. coli, was described 60 . Addition of


Ž .His6 tag to the Hepatitis B virus core antigen HBcAg , expressed inE. coli, enabledpurification under milder denaturing conditions by Ni–NTA at high pH. ContaminatingE. coli proteins and DNA were completely removed. This was otherwise impossible

w xby standard sedimentation of virus-like particles in sucrose gradients 43 . Whole chi-Ž .meric virus-like particles of infectious bursal disease young-chicken virus disease

Ž .were isolated on Ni II ProBonde from insect cells, Sf-9, coinfected with twostrains of baculoviruses. To one protein a His5 tag was added, which ensured suf-ficiently strong binding to the IMAC matrix and mild elution of particles. Thisapproach avoided extensive centrifugation and led to simple and low-cost vaccine

w xproduction 61 .A malaria-transmission-blocking vaccine candidate, based on thePlasmodium falci-

parum predominant surface protein Pfs25 with a His6-tag at the carboxyl terminus, wasproduced by secretion fromSaccharomyces cereÕisiae and purified on a large scale byNi–NTA. Histidine-tagged protein exhibited higher potency and antigenicity than the

w xoriginal Pfs25 45 . This indicates that in some cases vaccination with His-taggedproteins may be even advantageous.

His6 tag was also used for producing several clinical-grade single-chain Fv antibodiesw x w x46,62 and IMAC proved superior to traditional antigen affinity chromatography 46 .

Ž .IMAC on Cu II -charged Chelating Sepharose was used for large-scale preparation ofw xclinical-grade factor IX 63 .

There are many more reports on the application of His6 tag for IMAC isolation ofpotential therapeutics, but the majority of them describe preliminary procedures, notusually giving details about histidine tag removal and final yields. However, IMACtechnology should be further improved with respect to metal-ion leakage, dynamiccapacity, reproducibility, etc. We can conclude that there are many attempts to useIMAC matrices for large-scale isolation of biopharmaceuticals, but many of them arestill in the trial phase, or else the data are not accessible to the public.

Ž .Expanded-bed adsorption EBA techniques constitute another broad field of IMACapplication and require additional properties of column matrix, e.g., higher particledensity and high resistance to harsh conditions during column cleaning or sanitization.Expanded-bed techniques are less attractive on a small, laboratory scale but potentiallyhighly advantageous at an industrial scale. Downstream processing procedures from

w xunclarified E. coli or yeast homogenates are being developed for native 32,64 as wellw xas histidine-tagged proteins 47 . Generally, recoveries over 80% of the protein were

achieved in successful cases, but at least two major weak features must be furtherŽ .improved: low dynamic capacity and efficiency of Clean In Place CIP procedures for

eliminating contaminants.Elimination of centrifugation and filtration in large industrial-scale isolations is a

major driving force for the introduction of EBA in the isolation of therapeutic proteins.Ž .Streamline Chelating Amersham Pharmacia Biotech has been tried to purify two

vaccine candidates for clinical studies: His6-tagged modified diphtheria toxin, expressedw xin E. coli, and malaria-transmission-blocking vaccine, secreted fromS. cereÕisiae 65 .

The combination of IMAC and EBA techniques should provide a unique approach tosimplifying the whole downstream process, reduce the number of steps and start-upinvestment, and thus make the purification more economical.


5. Some other applications of IMAC

IMAC has become very popular, especially for scientific or research work on alaboratory scale. Commercial cloning vectors, containing sequences that encode histi-

Ž .dine tags, are available as well as antibodies Table 2 for specific detection ofw xHis-tagged proteins 66 . For rapid high-throughput laboratory detection or purification

of His6-tagged recombinant proteins from cleared lysates on 96-well plates, differentsystems were developed. For immobilization and subsequent analytical purposes, a low

Ž .capacity of up to 0.25mg proteinrwell, e.g., Ni–NTA HisSorb Strips Qiagen , isŽ .sufficient, while high-capacity systems, e.g., SwellGele Nickel Chelated Discs Pierce ,

offer isolation of up to 1 mg proteinrwell, to name just two examples. New varieties arecoming out rapidly, focused on automatic processing by robots for handling largeensembles of proteins, as is the case in functional genomics, engineered enzymes,

Ž .antibody and drug screening. In Biacore instruments Biacore, Uppsala , which aredesigned to study protein interactions, the sensor chip covered with Ni–NTA has beenused for simple attachment of histidine-tagged proteins . For instance, His-tagged GroESwas immobilized and interactions with GroEL under various conditions were studiedw x67 . This approach can be applied to studying interactions in many protein complexes,provided that one of the proteins carries a histidine tag not interfering with its biologicalactivity.

In proteins containing no histidines, which bind to IMAC at high pH due to theaccessible N-terminala-amino group, this type of chromatography can be used to reveal

w xmodifications of the N-terminal 1 . IMAC has also been used for affinity purification ofnonprotein molecules, such as DNA, by employing an affinity tag of six successive6-histaminylpurine residues, which mediate selective adsorption to Ni–NTA chelate

w xresin 68 . Native DNA binds weakly to IMAC matrices, while RNA and oligonucleo-w xtides bind strongly due to accessible aromatic nitrogens in the bases 69 .

In the last decade, some nonchromatographic techniques have appeared, such asMetal-Affinity Precipitation of proteins with attached histidine affinity tails through

Ž . w xformation of a metal chelate complex, e.g., with EGTA Zn 70 or with new2Ž . w xCu II -loaded copolymers 71 . Immobilized-Metal-Ion Affinity Partitioning is another

related technique for preparative extraction of proteins based on different content anddistribution of histidine residues. Aqueous two-phase systems containing Metal–IDA–PEG in PEG–dextran and PEG–salt systems have been used not only for extracting

w x w xproteins 72 but also for affinity partitioning of human blood cells like erythrocytes 73w xor lymphocytes 74 .

Ž .Immobilized-Metal-Ion Affinity Electrophoresis on, e.g., PEG–IDA–Cu II in agarosew xgels 75 , and Immobilized-Metal-Ion Affinity Capillary Electrophoresis with soluble-

w xpolymer-supported ligands 76 are examples of further applications of the same basicprinciple, although none of these techniques has become as popular as IMAC itself.

Another perspective separation technique emerged a few years ago, combining metalŽaffinity and magnetic separation. Commercial ferromagnetic Dynabeads M-280 Dynal,

. Ž .Lake Success were derivatized with NTA and charged with Ni II . Specific binding andw xelution of small radiolabelled His6-Ala-Tyr-Gly peptide was demonstrated 77 , indicat-

ing the potential for rapid isolation of His-tagged or native histidine-rich proteins.


Similarly, IDA-derivatized magnetic adsorbent, based on BioMag superparamagneticŽ .particles Perseptive Diagnostics, Cambridge, MA , was applied for a single-step, batch

w xisolation of histidine-tailed T4 lysozyme from crudeE. coli extracts 78 . In anotherŽ . Ž .case, IDA–Cu II or IDA–Zn II magnetic agarose was used to study separation of

w xmodel protein from E. coli cell lysate 79 . At least one commercial kit, based onŽ .Ni–NTA Magnetic Agaraose Beads Qiagen has recently been introduced for high-

throughput, micro-scale purification of His6-tagged proteins on 96-well plates. Powerfulmagnetic rods fit between the wells in such a way that beads are firmly kept on the wallsduring washing, elution or exchange of buffers. The system seems useful for screeningpurposes as well as for concentrating poorly expressed proteins followed by variousassay procedures.

6. Some advantages of IMAC

6.1. Use under denaturing conditions and in situ refolding

Today, many recombinant proteins are produced by intracellular expression ofheterologous genes in genetically engineeredE. coli strains. However, recombinantproteins, accumulated intracellularly, are frequently deposited in the form of insolubleaggregates of misfolded proteins in inclusion bodies. The production of pure, biologi-cally active proteins involves denaturation and refoldingrrenaturation, which is classi-cally accomplished by the low-efficiency techniques of dialysis or dilution. IMACchromatography has the advantage of enabling histidine-tagged proteins to be separatedefficiently in the presence of denaturing concentrations of urea or guanidine–HCl.Additionally, affinity tagging by consecutive histidines offers the possibility of efficient

w xpurification and refolding in a single IMAC step. GATA-1 protein 80 , antigens ofw x w xMycobacterium tuberculosus 81 , and two different membrane proteins 82 are some

examples of His-tagged fusion proteins, purified under denaturing conditions, renaturedwhile still bound to the solid phase by lowering the concentration of chaotropic agentand then eluted from IMAC columns in the renatured, biologically active form.

6.2. Immobilization on the column

Due to the relatively high affinity of histidine tags for special IMAC matrices thesecan serve for the reversible immobilization of proteins. An interesting example is theisolation of the multimeric chaperonin GroEL, containing His6 tail on the C-terminus,from a crude extract of recombinantE. coli and its subsequent immobilization onNi-chelate Cellulofine. The protein retained its ability to mediate protein refolding when

w xattached to the affinity chelating matrix 83 . Similarly, IMAC matrices were used forw x w xthe reversible immobilization of 2-haloacid dehalogenase 84 andb-galactosidase 85 ,

which retained their enzymatic activities. As the affinity of the His6-tagged proteins forNi–NTA matrix is higher than the affinity between most antibodies and antigens, the


recombinant protein can be immobilized on such columns and serve for the affinityw xpurification of specific antibodies 86 .

6.3. Use of detergents and organic solÕents

Although consecutive histidines sequences are not found in natural proteins, andalthough interactions of engineered oligo-histidine tags with metal-chelated ligands arethought to be highly specific, protein and nonprotein contaminants can bind nonspecifi-cally to the chromatographic matrices or to the tagged protein itself. In this respect,IMAC matrices have the advantage of enabling the use of relatively harsh conditions.

w xThus, with membrane proteins, detergents can be used 42 . Especially when thehistidine tail is extended to 10 residues, stringent washes at high imidazol concentrationsresult in more efficient removal of host cell proteins. IMAC columns are relativelyefficient for virus removal, and reduction of 4.0 log to 6.5 log for different viruses has

w xbeen demonstrated during Co-chelate chromatography of factor IX 87 . Removal ofendotoxins can be significantly improved by a column wash with organic solvents, e.g.,

w x60% 2-propanol 88 . Employment of His10 tags in the case of trimeric TNF-a alsoresulted in efficient host-cell endotoxin and DNA removal, although some residualE.

w xcoli proteins are simultaneously eluted during the final EDTA elution 89 .

7. Some disadvantages and problems of IMAC

7.1. OxidatiÕe reductiÕe conditions inside the column and metal-induced cleaÕage

Several amino acids, especially histidine, lysine, cysteine, proline, arginine, andmethionine, are susceptible to metal-catalyzed oxidation reactions that produce highly

w xreactive radical intermediates which can damage a variety of proteins 90 . Taking intoŽ .account that metal chelates as well as Cu II ions themselves can be used for the

w xsite-specific cleavage of proteins 91–93 , it is not surprising that destruction of aminoacid side chains and cleavage of the protein backbone can also be provoked during

Ž .IMAC chromatography. In such cases, the replacement of high redox-active Cu II withŽ .a less active metal ion, such as Zn II , may prevent, or at least minimize, protein damage

w x90 .The majority of routine IMAC separations are carried out under aerobic, mildly

oxidative conditions, due to oxygen dissolved in the sample and buffers. Potentialdamage to proteins, caused by reactive oxygen species or metal-catalyzed reactionsinside the IMAC column, has not been studied enough. In experiments under forcedconditions, e.g., when hydrogen peroxide or ascorbate—and especially a combination ofboth—were added to elution buffers, a significant loss of protein activity was demon-

Ž . w xstrated on Cu II -IDA columns 90 . This was ascribed to reactions of hydroxyl radicals,Ž nq P y Žnq1.q.which are produced by the Fenton reaction MqH O ™OH qOH qM .2 2

Experiments with short model peptides confirmed that histidines react readily in this


w xtype of reaction 94 . Thus, in real IMAC columns, the potential for oxidative radicaldamage exists, especially in cases where extensive elution with widely used detergents,such as Tween 80 or Triton X-100, contaminated with hydroperoxy compounds, isperformed. Low in-column contact times and use of superflow matrices would bepreferable in such cases.

When IMAC is used under denaturing conditions, reducing agents, such asb-mer-Ž . Ž .captoethanolb-ME or dithiothreitol DTT , are usually added to the buffers used to

dissolve inclusion bodies and also during chromatography to prevent formation ofdisulfide-bonded aggregates. With some IMAC matrices, low concentrations of reducingagents, usuallyb-ME in the millimolar range, are permitted, although the columncapacity is rapidly diminished and chromatographic performance becomes irreproducibleafter several applications of the sample. In this respect, TALON with immobilized

Ž .Co II should be the matrix of choice, since, according to the manufacturer, it toleratesw xup to 30 mMb-ME under native conditions 95 . Although IMAC was also used for the

w xresolution of sulfitolyzed proteins 96 , no data exist for the highest concentrations ofsulfite allowed.

7.2. Metal toxicity

With every IMAC column some leaching of metal ions occurs, depending on the typeof chelating compound involved and the sort of elution. In this respect, tetradentatechelators, such as Ni–NTA or TALON, are superior to tridentates. Ni–NTA was stated

w xto exhibit very low leaching in the range of up to 1 ppm 97 . Nevertheless, usingŽ .Ni–NTA agarose, contamination with about 2 mol of Ni II per mole of His6-tagged

w xrestriction endonucleaseEcoRV has been demonstrated 98 .Ž . w x Ž .Ni II compounds are established human carcinogens 99 . Although the role of Ni II

in carcinogenesis is not clear, some molecular models suggest interaction with histonesin the cell nucleus, leading to DNA damage. Analyses of a synthetic oligopeptide,imitating the N-terminal part of human H4 histone, have confirmed that histidine acts as

Ž . w xa metal-binding site and is most probably responsible for Ni II toxicity 100 .Purification of pharmaceutical proteins demands special caution, since the possibilityŽ .of Ni II or other metal ions being trapped in the protein interior could result in higher

uptake by the target cells, through internalization mechanisms via receptors, especiallyin the case of long-term therapy. For safety reasons and regulatory requirements,analyses of pure protein are needed to prove that no incorporation of metal ions into theprotein has occurred. A structurally captured metal ion is most probably more dangerousthan metal ions resulting from leaching, which can be dealt with by different ap-proaches, e.g., diafiltration versus EDTA containing buffers.

Long-term stability of the final formulation can be significantly reduced if capturedmetal ions slowly difuse into the surrounding medium, where they can act as catalystsfor cleavage reactions. Removal of residual or adsorbed metal ions can be achieved by

w xre-chromatography on noncharged IMAC columns 7,8,98 or by the addition ofw xchelating agents, such as EDTA, to the collecting vials 7 . In addition, in the

purification strategy IMAC is usually the first chromatographic step, followed by severalpolishing steps.


7.3. RemoÕal of histidine tags

Polyhistidine extensions usually do not compromise the biological protein function.Histidine chains are not very immunogenic, and there are some examples where newlyintroduced or attached histidines do not interfere with potential therapeutic use. Thus, aHis6-bearing malaria vaccine peptide was reported not to elicit production of antibodies

w xin animal models 60 . In pre-clinical testing on mice, a double-histidine mutant ofTNF-a showed lower systemic toxicity than the natural counterpart and served as a leadcompound for the design of novel analogs with improved properties for cancer therapyw x101 . Additionally, several examples of the use of His6-tagged proteins for clinical

w xtrials are known 45,46 . However, for most pharmaceutical-grade proteins the authenticstructure is usually required and the affinity tags have to be chemically or enzymaticallyremoved. Chemical cleavage involving protein-destroying conditions and toxic chemi-cals is usually undesirable. Another approach is to apply proteolytic enzymes, such as

w xcoagulation factor Xa, thrombin, enterokinase or carboxypeptidase 102 that recognizespecific amino acid sequences. Although many proteases are declared to cleave specifi-cally, their efficiency depends on the accessibility of the cleavage site and adjacentresidues. Enterokinase was reported to be an ideal cleavage enzyme with cleavageefficiencies from 60% to almost 90%, depending on the first amino acid residue

w xdownstream of the recognition sequence 103 . However, when N-terminal oligo-histi-dine tags on TNF-a were removed by enterokinase, nonspecific cleavage also occurred,

Ž .resulting in micro-heterogeneity of the N-terminus Gaberc-Porekar, unpublished results .This is not acceptable for pharmaceutical-grade proteins. Commercial recombinant

Ž .His-tagged rTEV Life Technologies , a protease from tobacco etch virus is available forw xreliable cleavage even atq4 8C 104, 105 and easy separation from the cleaved protein.

However, as cleavage occurs between Gln-Gly residues, an additional Gly remainsattached to the N-terminus of the protein which is usually undesirable for therapeuticproteins, where authentic termini are requested. Recently, some new endoproteases wereintroduced which offer certain advantages. Protease 3C from a picornavirus has im-proved specificity of cleavage and was designed for easy one-step removal of both the

w xprotease and the affinity tail 106 . A kallikrein-like protease from snake venom hasbeen used to cleave a polyhistidine tag, but general application of this system has yet to

w xbe explored 107 . A possible alternative is the use of one or three exopeptidases,catalyzing a stepwise cleavage of dipeptides from the N-terminus of specially designed

w xhistidine tags 108 . The additional benefit of this system is that the enzymes used areengineered to bind to the IMAC matrices by themselves, and this enables easy andeconomical removal. Cleavage of His-tag from the C-terminus is rarely reported

w xalthough it is possible by using carboxypeptidase A 70,109 . In many cases, afterenzymatic cleavage of the fusion protein, removal of the enzyme, uncleaved protein, andany other cleavage products remains a problem, especially in the case of therapeuticproteins, where stringent GMP criteria apply.

7.4. Miscellaneous

Perhaps the greatest value of His6-tagged protein purification stems from the factthat, in most instances, His6 tags do not affect protein activity. However, a recent paper,


w xdescribing His6-tagged-dependent protein dimerization 110 , sends an important mes-sage that other His-tagged proteins may differ from their authentic counterparts incertain properties. In the case of single chain monoclonal antibody CC49, extensivelystudied for cancer therapy, addition of hexahistidine tag to the C-terminus substantially

w xreduced affinity towards the antigen 111 . On the other hand, His6 tag added to theŽ .C-terminus of heparin cofactor II HC II , increased the inhibition of thrombin more

than twofold. When His tag was removed by carboxypeptidase A treatment, anti-coagulant activity was again comparable to that of the wild type recombinant HC IIw x109 . The addition of His tags may result in a lower expression level of the taggedprotein compared to the untagged protein. This was noted in the case of TNF-a , where

w xaccumulation inE. coli was reduced up to threefold for a His10–TNF 52 .The reported capacities of the commercial IMAC sorbents are usually in the range of

5–10 mgrml or even higher. These values refer to isolated pure proteins or syntheticmixtures, while capacities for isolation of the target protein from complex sources areusually lower. Some matrices, e.g., those based on IDA chelator, allow a large numberof cycles of charging the column with metal ions and stripping them off. For morestrongly bound metals, such as in the case with Co–TALON or Ni–NTA, the suppliersdo not recommend stripping off and recharging. This is not very attractive for commer-cial applications, due to high cost and frequent labor of packing large columns.

SinceAhistidine ligand chromatographyB, in which immobilized histidine functions asa pseudoaffinity tag, can be used for selective binding of lipo-polysaccharides and

w xeffective pyrogen removal 112 , it is reasonable to assume that histidine affinity tagscould also bind pyrogens. Thus, contamination of the target protein with pyrogens wouldoccur, and this is not acceptable for pharmaceutical proteins. However, our recentexperiments with His10-tagged TNF-a , isolated fromE. coli homogenates on Chelating

Ž . w xSepharose Amersham Pharmacia Biotech , have shown 89 that the target proteineluted by EDTA contains very low amounts of endotoxins, below the allowable limitsfor clinical-grade proteins.

8. Conclusion

Taken all together, almost all of Porath’s and Sulkowski’s visions and forecasts ofw xmany years ago 3,8 have come true. In the meantime, engineered histidine affinity tags

have appeared and IMAC has become a popular tool for easy isolation of recombinantproteins, used in almost every laboratory. However, in the era of biopharmaceuticals,there are some dilemmas and problems that should also be considered when IMACtechnologies are to be used for the production of clinical-grade proteins.

Acknowledgements

We thank Prof. Roger Pain for careful reading of the manuscript and usefulsuggestions. Dr. Lothar Jacob is acknowledged for providing templates used in Fig. 2.


References

w x1 Arnold FH. Metal-affinity separations: a new dimension in protein processing. BiorTechnology1991;9:150–5.

w x2 Everson RJ, Parker HE. Zinc binding and synthesis eight-hydroxy-quinoline-agarose. Bioinorg Chem1974;4:15–20.

w x3 Porath J, Carlsson J, Olsson I, Belfrage G. Metal chelate affinity chromatography, a new approach toprotein fractionation. Nature 1975;258:598–9.

w x4 Porath J, Olin B. Immobilized metal ion affinity adsorption and immobilized metal ion affinitychromatography of biomaterials: serum protein affinities for gel-immobilized iron and nickel ions.Biochemistry 1983;22:1621–30.

w x5 Ramadan N, Porath J. Fe3q-hydroxamate as immobilized metal affinity-adsorbent for protein chro-matography. J Chromatogr 1985;321:93–104.

w x6 Hemdan ES, Zhao YJ, Sulkowski E, Porath J. Surface topography of histidine residues: a facile probe byimmobilized metal ion affinity chromatography. Proc Natl Acad Sci U S A 1989;86:1811–5.

w x7 Porath J. Immobilized metal ion affinity chromatography. Protein Expression Purif 1992;3:263–81.w x8 Sulkowski E. Purification of Proteins by IMAC. Trends Biotechnol 1985;3:1–7.w x9 Sulkowski E. The saga of IMAC and MIT. Bioessays 1989;10:170–5.w x10 Sulkowski E. Immobilized metal-ion affinity chromatography: imidazole proton pump and chromato-

graphic sequelae: I. Proton pump. J Mol Recognit 1996;9:389–93.w x11 Sulkowski E. Immobilized metal-ion affinity chromatography: imidazole proton pump and chromato-

graphic sequelae: II. Chromatographic sequelae. J Mol Recognit 1996;9:494–8.w x12 Sulkowski E. Immobilized metal ion affinity chromatography of proteins. In: Burgess R, editor. Protein

purification, micro to macro. New York: Allan R. Liss; 1987. p. 149–62.w x13 Hochuli E, Doebeli H, Schacher A. New metal chelate adsorbent selective for proteins and peptides

containing neighbouring histidine residues. J Chromatogr 1987;411:177–84.w x14 Hochuli E, Bannwarth W, Doebeli H, Gentz R, Stueber D. Genetic approach to facilitate purification of

recombinant proteins with a novel metal chelate adsorbent. BiorTechnology; 1988. p. 1321–5.w x15 Yip TT, Hutchens TW. Immobilized metal ion affinity chromatography. Mol Biotechnol 1994;1:151–64.w x16 Johnson RD, Arnold FH. Multipoint binding and heterogeneity in immobilized metal affinity chro-

matography. Biotechnol Bioeng 1995;48:437–43.w x17 Liesiene J, Racaityte K, Morkeviciene M, Valancius P, Bumelis V. Immobilized metal affinity

chromatography of human growth hormone—effect of ligand density. J Chromatogr, A 1997;764:27–33.w x18 Zachariou M, Hearn MT. Protein selectivity in immobilized metal affinity chromatography based on the

surface accessibility of aspartic and glutamic acid residues. J Protein Chem 1995;14:419–30.w x19 Reif O-W, Nier V, Bahr U, Freitag R. Immobilized metal affinity membrane adsorbers as stationary

phases for metal interaction protein separation. J Chromatogr, A 1994;664:13–25.w x20 Serafica GC, Pimbley J, Belfort G. Protein fractionation using fast flow immobilized metal chelate

affinity membranes. Biotechnol Bioeng 1994;43:21–36.w x21 Kubota N, Nakagawa Y, Eguchi Y. Recovery of serum proteins using cellulosic affinity membrane

modified by immobilization of Cu2q ion. J Appl Polym Sci 1996;62:1153–60.w x22 Crawford J, Ramakrishnan S, Periera P, Gardner S, Coleman M, Beitle R. Immobilized metal affinity

membrane separation: characteristics of two materials of differing preparation chemistries. Sep SciTechnol 1999;34:2793–802.

w x23 Camperi SA, Grasselli M, Cascone O. High-speed pectic enzyme fractionation by immobilised metal ionaffinity membranes. Bioseparation 2000;9:173–7.

w x Ž .24 Arica MY, Testereci HN, Denizli A. Dye-ligand and metal chelate poly 2-hydroxyethylmethacrylatemembranes for affinity separation of proteins. J Chromatogr, A 1998;799:83–91.

w x Ž25 Arica MY, Denizli A, Baran T, Hasirci V. Dye derived and metal incorporated affinity poly 2-hydroxy-.ethylmetacrylate membranes for use in enzyme immobilization. Polym Int 1998;46:345–52.

w x26 Hari PR, Paul W, Sharma CP. Adsorption of human IgG on Cu2q-immobilized cellulose affinitymembrane: preliminary study. J Biomed Mater Res 2000;50:110–3.


w x27 Xiao J, Meyerhoff ME. Retention behavior of amino acids and peptides on protoporphyrin-silicastationary phases with varying metal ion centers. Anal Chem 1996;68:2818–25.

w x28 Wu HP, Bruley DF. Homologous human blood protein separation using immobilized metal affinitychromatography: protein C separation from prothrombin with application to the separation of factor IXand prothrombin. Biotechnol Prog 1999;15:928–31.

w x29 Dodd I, Jalalpour S, Southwick W, Newsome P, Browne MJ, Robinson JH. Large scale, rapidpurification of recombinant tissue-type plasminogen activator. Febs Lett 1986;209:13–7.

w x30 Boden V, Winzerling JJ, Vijayalakshmi M, Porath J. Rapid one-step purification of goat immuno-globulins by immobilized metal ion affinity chromatography. J Immunol Methods 1995;181:225–32.

w x31 Freyre FM, Vazquez JE, Ayala M, Canaan-Haden L, Bell H, Rodriguez I, et al. Very high expression ofan anti-carcinoembryonic antigen single chain Fv antibody fragment in the yeastPichia pastoris. JBiotechnol 2000;76:157–63.

w x32 Willoughby NA, Kirschner T, Smith MP, Hjorth R, Titchener-Hooker NJ. Immobilised metal ionaffinity chromatography purification of alcohol dehydrogenase from baker’s yeast using an expandedbed adsorption system. J Chromatogr, A 1999;840:195–204.

w x33 Sagar SL, Beitle RR, Ataai MM, Domach MM. Metal-based affinity separation of alpha- andgamma-chymotrypsin and thermal stability analysis of isolates. Bioseparation 1992;3:291–6.

w x34 Zhao YJ, Sulkowski E, Porath J. Surface topography of histidine residues in lysozymes. Eur J Biochem1991;202:1115–9.

w x Ž .35 Chaouk H, Hearn MT. New ligand,N- 2-pyridylmethyl aminoacetate, for use in the immobilised metalion affinity chromatographic separation of proteins. J Chromatogr, A 1999;852:105–15.

w x Ž .36 Chaouk H, Hearn MT. Examination of the protein binding behaviour of immobilised copper II -2,6-di-aminomethylpyridine and its application in the immobilised metal ion affinity chromatographic separa-tion of several human serum proteins. J Biochem Biophys Methods 1999;39:161–77.

w x37 Beitle RR, Ataai MM. One-step purification of a model periplasmic protein from inclusion bodies by itsfusion to an effective metal-binding peptide. Biotechnol Prog 1993;9:64–9.

w x38 Chaga G, Hopp J, Nelson P. Immobilized metal ion affinity chromatography on Co2q-carboxy-methylaspartate-agarose Superflow, as demonstrated by one-step purification of lactate dehydrogenase

Ž .from chicken breast muscle. Biotechnol Appl Biochem 1999;29 Pt 1 :19–24.w x39 Chaga G, Bochkariov DE, Jokhadze GG, Hopp J, Nelson P. Natural poly-histidine affinity tag for

Ž .purification of recombinant proteins on cobalt II -carboxymethylaspartate crosslinked agarose. J Chro-matogr, A 1999;864:247–56.

w x40 Smith MC, Furman TC, Ingolia TD, Pidgeon C. Chelating peptide-immobilized metal ion affinitychromatography: a new concept in affinity chromatography for recombinant proteins. J Biol Chem1988;263:7211–5.

w x41 Ljungquist C, Breitholtz A, Brink-Nilsson H, Moks T, Uhlen M, Nilsson B. Immobilization and affinitypurification of recombinant proteins using histidine peptide fusions. Eur J Biochem 1989;186:563–9.

w x42 Grisshammer R, Tucker J. Quantitative evaluation of neurotensin receptor purification by immobilizedmetal affinity chromatography. Protein Expression Purif 1997;11:53–60.

w x43 Wizemann H, von Brunn A. Purification ofE. coli-expressed His-tagged hepatitis B core antigen byNi2q-chelate affinity chromatography. J Virol Methods 1999;77:189–97.

w x44 Cha HJ, Wu CF, Valdes JJ, Rao G, Bentley WE. Observations of green fluorescent protein as a fusionpartner in genetically engineeredEscherichia coli: monitoring protein expression and solubility.Biotechnol Bioeng 2000;67:565–74.

w x45 Kaslow DC, Shiloach J. Production, purification and immunogenicity of a malaria transmission-blockingvaccine candidate: TBV25H expressed in yeast and purified using nickel–NTA agarose. BiotechnologyŽ .N Y 1994;12:494–9.

w x46 Casey JL, Keep PA, Chester KA, Robson L, Hawkins RE, Begent RH. Purification of bacteriallyexpressed single chain Fv antibodies for clinical applications using metal chelate chromatography. JImmunol Methods 1995;179:105–16.

w x47 Clemmitt RH, Chase HA. Facilitated downstream processing of a histidine-tagged protein fromunclarified E. coli homogenates using immobilized metal affinity expanded-bed adsorption. BiotechnolBioeng 2000;67:206–16.


w x48 Wu CF, Cha HJ, Rao G, Valdes JJ, Bentley WE. A green fluorescent protein fusion strategy formonitoring the expression, cellular location, and separation of biologically active organophosphorushydrolase. Appl Microbiol Biotechnol 2000;54:78–83.

w x49 Goud GN, Patwardhan AV, Beckman EJ, Ataai MM, Koepsel RR. Selection of specific peptide ligandsfor immobilised metals using a phage displayed library: application to protein separation using IMAC.IJBC 1997;3:123–36.

w x50 Patwardhan AV, Goud GN, Koepsel RR, Ataai MM. Selection of optimum affinity tags from aŽ .phage-displayed peptide library: application to immobilized copper II affinity chromatography. J

Chromatogr, A 1997;787:91–100.w x51 Pasquinelli RS, Shepherd RE, Koepsel RR, Zhao A, Ataai MM. Design of affinity tags for one-step

protein purification from immobilized zinc columns. Biotechnol Prog 2000;16:86–91.ˇw x52 Gaberc-Porekar V, Menart V, Jevsevar S, Vidensek A, Stalc A. Histidines in affinity tags and surfaceˇ ˇ

clusters for immobilized metal-ion affinity chromatography of trimeric tumor necrosis factor alpha. JChromatogr, A 1999;852:117–28.

w x53 Armisen P, Mateo C, Cortes E, Barredo JL, Salto F, Diez B, et al. Selective adsorption of poly-Histagged glutaryl acylase on tailor-made metal chelate supports. J Chromatogr, A 1999;848:61–70.

w x54 Seidler A. Introduction of a histidine tail at the N-terminus of a secretory protein expressed inEscherichia coli. Protein Eng 1994;7:1277–80.

w x Ž .55 Todd RJ, Van Dam ME, Casimiro D, Haymore BL, Arnold FH. Cu II -binding properties of acytochromec with a synthetic metal-binding site: His-X3-His in an alpha-helix. Proteins 1991;10:156–61.

w x Ž .56 Muller HN, Skerra A. Grafting of a high-affinity Zn II -binding site on the beta- barrel of retinol-bind-¨ing protein results in enhanced folding stability and enables simplified purification. Biochemistry1994;33:14126–35.

w x Ž .57 Yilmaz S, Widersten M, Emahazion T, Mannervik B. Generation of a Ni II binding site by introductionof a histidine cluster in the structure of human glutathione transferase A1-1. Protein Eng 1995;8:1163–9.

w x58 Chaga G, Widersten M, Andersson L, Porath J, Danielson UH, Mannervik B. Engineering of a metalcoordinating site into human glutathione transferase M1-1 based on immobilized metal ion affinitychromatography of homologous rat enzymes. Protein Eng 1994;7:1115–9.

w x59 Menart V, Gaberc-Porekar V, Harb V. Metal-affinity separation of model proteins having differentlyspaced clusters of histidine residues. In: Pyle DL, editor. Separations for biotechnology, vol. 3,Cambridge: The Royal Society of Chemistry; 1994. p. 308–13.

w x60 Takacs BJ, Girard MF. Preparation of clinical grade proteins produced by recombinant DNA technolo-gies. J Immunol Methods 1991;143:231–40.

w x61 Hu YC, Bentley WE, Edwards GH, Vakharia VN. Chimeric infectious bursal disease virus-like particlesexpressed in insect cells and purified by immobilized metal affinity chromatography. Biotechnol Bioeng1999;63:721–9.

w x62 Laroche-Traineau J, Clofent-Sanchez G, Santarelli X. Three-step purification of bacterially expressedhuman single-chain Fv antibodies for clinical applications. J Chromatogr, B: Biomed Sci Appl 2000;737:107–17.

w x63 Feldman PA, Bradbury PI, Williams JD, Sims GE, Mcphee JW, Pinnell MA, et al. Large-scalepreparation and biochemical characterization of a new high purity factor IX concentrate prepared bymetal chelate affinity chromatography. Blood Coagulation Fibrinolysis 1994;5:939–48.

w x64 Clemmitt RH, Chase HA. Immobilised metal affinity chromatography of beta-galactosidase fromunclarified Escherichia coli homogenates using expanded bed adsorption. J Chromatogr, A2000;874:27–43.

w x65 Noronha S, Kaufman J, Shiloach J. Use of streamline chelating for capture and purification ofpoly-His-tagged recombinant proteins. Bioseparation 1999;8:145–51.

w x66 Lindner P, Bauer K, Krebber A, Nieba L, Kremmer E, Krebber C, et al. Specific detection of his-taggedproteins with recombinant anti-His tag scFv-phosphatase or scFv-phage fusions. Biotechniques1997;22:140–9.

w x67 Nieba L, Nieba-Axmann SE, Persson A, Hamalainen M, Edebratt F, Hansson A, et al. BIACORE¨ ¨ ¨analysis of histidine-tagged proteins using a chelating NTA sensor chip. Anal Biochem 1997;252:217–28.


w x68 Min C, Verdine GL. Immobilized metal affinity chromatography of DNA. Nucleic Acids Res1996;24:3806–10.

w x69 Murphy JC, White KI, Willson RC. Nucleic acid separation using immobilized metal affinity chro-matography. Abstract BIOT 347—219th ACS national meeting in San Francisco—March 26–30, 2000.

w x70 Lilius G, Persson M, Bulow L, Mosbach K. Metal affinity precipitation of proteins carrying geneticallyattached polyhistidine affinity tails. Eur J Biochem 1991;198:499–504.

w x71 Mattiasson B, Kumar A, Galaev IY. Affinity precipitation of proteins: design criteria for an efficientpolymer. J Mol Recognit 1998;11:211–6.

w x72 Otto A, Birkenmeier G. Recognition and separation of isoenzymes by metal chelates. Immobilized metalion affinity partitioning of lactate dehydrogenase isoenzymes. J Chromatogr 1993;644:25–33.

w x73 Botros HG, Birkenmeier G, Otto A, Kopperschlager G, Vijayalakshmi MA. Immobilized metal ionaffinity partitioning of cells in aqueous two-phase systems: erythrocytes as a model. Biochim BiophysActa 1991;1074:69–73.

w x74 Laboureau E, Capiod JC, Dessaint C, Prin L, Vijayalakshmi MA. Study of human cord bloodlymphocytes by immobilized metal ion affinity partitioning. J Chromatogr, B: Biomed Appl 1996;680:189–95.

w x75 Goubran-Botros H, Nanak E, Abdul N, Birkenmeir G, Vijayalakshmi MA. Immobilized metal ionaffinity electrophoresis: a study with several model proteins containing histidine. J Chromatogr1992;597:357–64.

w x76 Haupt K, Roy F, Vijayalakshmi MA. Immobilized metal ion affinity capillary electrophoresis of proteins—a model for affinity capillary electrophoresis using soluble polymer-supported ligands. Anal Biochem1996;234:149–54.

w x77 Ji Z, Pinon DI, Miller LJ. Development of magnetic beads for rapid and efficient metal-chelate affinitypurifications. Anal Biochem 1996;240:197–201.

w x78 O’Brien SM, Sloane RP, Thomas OR, Dunnill P. Characterisation of non-porous magnetic chelatorsupports and their use to recover polyhistidine-tailed T4 lysozyme from a crudeE. coli extract. JBiotechnol 1997;54:53–67.

w x79 Abudiab T, Beitle RR. Preparation of magnetic immobilized metal affinity separation media and its usein the isolation of proteins. J Chromatogr, A 1998;795:211–7.

w x80 Apezteguia I, Calligaris R, Bottardi S, Santoro C. Expression, purification, and functional characteriza-tion of the two zinc-finger domain of the human GATA-1. Protein Expression Purif 1994;5:541–6.

w x81 Colangeli R, Heijbel A, Williams AM, Manca C, Chan J, Lyashchenko K, et al. Three-step purificationof lipopolysaccharide-free, polyhistidine-tagged recombinant antigens ofMycobacterium tuberculosis. JChromatogr, B: Biomed Sci Appl 1998;714:223–35.

w x82 Rogl H, Kosemund K, Kuhlbrandt W, Collinson I. Refolding ofEscherichia coli produced membrane¨protein inclusion bodies immobilised by nickel chelating chromatography. Febs Lett 1998;432:21–6.

w x83 Teshima T, Mashimo S, Kondo A, Fukuda H. Affinity purification and immobilization of fusionŽ .Ž .chaperonin GroEL His 6 and its utilization to mediate protein refolding. J Ferment Bioeng 1998;86:

357–62.w x84 Ordaz E, Garrido-Pertierra A, Gallego M, Puyet A. Covalent and metal-chelate immobilization of a

modified 2-haloacid dehalogenase for the enzymatic resolution of optically active chloropropionic acid.Biotechnol Prog 2000;16:287–91.

w x85 Brena BM, Ryden LG, Porath J. Immobilization ofb-galactosidase on metal-chelate-substituted gels.Biotechnol Appl Biochem 1994;17:217–31.

w x86 Gu J, Stephenson CG, Iadarola MJ. Recombinant proteins attached to a nickel–NTA column: use inaffinity purification of antibodies. Biotechniques 1994;17:257, 260, 262.

w x87 Roberts PL, Walker CP, Feldman PA. Removal and inactivation of enveloped and non-envelopedviruses during the purification of a high-purity factor IX by metal chelate affinity chromatography. Vox

Ž .Sang 1994;67 Suppl. 1 :69–71.w x88 Franken KLMC, Hiemstra HS, vanMeijgaarden KE, Subronto Y, denHartigh J, Ottenhoff THM, et al.

Purification of His-tagged proteins by immobilized chelate affinity chromatography: the benefits fromthe use of organic solvent. Protein Expression Purif 2000;18:95–9.

w x89 Menart V, Gaberc-Porekar V. Use of long histidine tags for efficient isolation of recombinant proteins.


Strancar, A. 20-20. 2000. Ljubljana, B.I.A. Book of abstracts: 20th international symposium on theseparation and analysis of proteins, peptides and polynucleotides. 5-11-2000.

w x90 Krishnamurthy R, Madurawe RD, Bush KD, Lumpkin JA. Conditions promoting metal-catalyzedoxidations during immobilized Cu-iminodiacetic acid metal affinity chromatography. Biotechnol Prog1995;11:643–50.

w x91 Rana TM. Artificial proteolysis by a metal chelate: methodology and mechanism. Adv Inorg Biochem1994;10:177–200.

w x92 Humphreys DP, Smith BJ, King LM, West SM, Reeks DG, Stephens PE. Efficient site specific removalX Ž .of a C-terminal FLAG fusion from a Fab using copper II ion catalysed protein cleavage. Protein Eng

1999;12:179–84.w x93 Cheng RZ, Kawakishi S. Site-specific oxidation of histidine residues in glycated insulin mediated by

Cu2q. Eur J Biochem 1994;223:759–64.w x94 Khossravi M, Borchardt RT. Chemical pathways of peptide degradation: IX. Metal-catalyzed oxidation

of histidine in model peptides. Pharm Res 1998;15:1096–102.w x95 Leo C, Nelson P, Kain S, Yang T. TALON resin offers 6xHis protein purification under native

conditions usingb-mercaptoethanol as a reducing agent. CLONTECHniques; 1996. p. 19.w x96 Kasher MS, Wakulchik M, Cook JA, Smith MC. One-step purification of recombinant human

papillomavirus type 16 E7 oncoprotein and its binding to the retinoblastoma gene product. Biotech-niques 1993;14:630–41.

w x97 Schafer F, Blumer J, Romer U, Steinert K. Ni–NTA for large-scale IMAC processes—systematic¨ ¨ ¨investigation of separation characteristics, storage and CIP conditions, and leaching. QIAGEN News;2000. p. 11–5.

w x Ž . w98 Oswald T, Hornbostel G, Rinas U, Anspach FB. Purification of His 6EcoRV recombinant restrictionŽ . xendonucleaseEcoRV fused to a His 6 affinity domain by metal-chelate affinity chromatography.Ž .Biotechnol Appl Biochem 1997;25 Pt 2 :109–15.

w x99 Bal W, Kozlowski H, Kasprzak KS. Molecular models in nickel carcinogenesis. J Inorg Biochem2000;79:213–8.

w x100 Zoroddu MA, Kowalik-Jankowska T, Kozlowski H, Molinari H, Salnikow K, Broday L, et al.Ž . Ž .Interaction of Ni II and Cu II with a metal binding sequence of histone H4: AKRHRK, a model of the

H4 tail. Bba Gen Subj 2000;1475:163–8.ˇ ˇw x101 Novakovic S, Menart V, Gaberc-Porekar V, Stalc A, Sersa G, Cemazar M, et al. New TNF-alpha´ ˇ ˇ

analogues: a powerful but less toxic biological tool against tumours. Cytokine 1997;9:597–604.w x102 Sassenfeld HM. Engineering proteins for purification. Trends Biotechnol 1990;8:88–93.w x Ž .Ž .103 Hosfield T, Lu Q. Influence of the amino acid residue downstream of Asp 4 Lys on enterokinase

cleavage of a fusion protein. Anal Biochem 1999;269:10–6.w x104 Leahy DJ, Dann III CE, Longo P, Perman B, Ramyar KX. A mammalian expression vector for

expression and purification of secreted proteins for structural studies. Protein Expression Purif 2000;20:500–6.

w x105 Pawelczyk T, Kowara R, Golebiowski F, Matecki A. Expression inEscherichia coli and simplepurification of human Fhit protein. Protein Expression Purif 2000;18:320–6.

w x106 Walker PA, Leong LE, Ng PW, Tan SH, Waller S, Murphy D, et al. Efficient and rapid affinityŽ .purification of proteins using recombinant fusion proteases. Biotechnology N Y 1994;12:601–5.

w x107 Hung CC, Chiou SH. Expression of a kallikrein-like protease from the snake venom: engineering ofautocatalytic site in the fusion protein to facilitate protein refolding. Biochem Biophys Res Commun2000;275:924–30.

w x108 Pedersen J, Lauritzen C, Madsen MT, Weis Dahl S. Removal of N-terminal polyhistidine tags fromrecombinant proteins using engineered aminopeptidases. Protein Expression Purif 1999;15:389–400.

w x109 Bauman SJ, Church FC. Enhancement of heparin cofactor II anticoagulant activity. J Biol Chem1999;274:34556–65.

w x Ž Ž ..110 Wu JW, Filutowicz M. Hexahistidine His 6 -tag dependent protein dimerization: a cautionary tale.Acta Biochim Pol 1999;46:591–9.

w x111 Goel A, Colcher D, Koo JS, Booth BJM, Pavlinkova G, Batra SK. Relative position of the hexahistidinetag effects binding properties of a tumor-associated single-chain Fv construct. Bba Gen Subj 2000;1523:13–20.


w x112 Vijayalakshmi MA. Histidine ligand affinity chromatography. Mol Biotechnol 1996;6:347–57.w x113 Leibler D, Rabinkov A, Wilchek M. Salicylaldehyde-metal-amino acid ternary complex: a new tool for

immobilized metal affinity chromatography. J Mol Recognit 1996;9:375–82.w x114 Zachariou M, Hearn MT. Application of immobilized metal ion chelate complexes as pseudocation

exchange adsorbents for protein separation. Biochemistry 1996;35:202–11.w x115 Zachariou M, Traverso I, Hearn MT. High-performance liquid chromatography of amino acids, peptides

and proteins: CXXXI. O-phosphoserine as a new chelating ligand for use with hard Lewis metal ions inthe immobilized-metal affinity chromatography of proteins. J Chromatogr 1993;646:107–20.

w x116 Nelson PS, Yang T-T, Kain SR. Method for purification of recombinant proteins. Patent US5962641,1999.

Perspectives of immobilized-metal affinity chromatography

Documents

Transcript of Perspectives of immobilized-metal affinity chromatography