Soluble Expression and Purification of Histidine-Tagged ...€¦ · While high concentration of...

17

Soluble Expression and Purification of Histidine-Tagged Moloney Murine

Leukemia Virus Reverse Transcriptase by Ni-NTA Affinity Chromatography

Xie Fei, Ma Xuemei and Wang Xiansong College of Life Science and Bio–Engineering, Beijing University of Technology, Beijing

PR China

1. Introduction

Purification of expressed proteins is often a time-consuming task and a limiting step in obtaining large quantities of proteins from crude cell extracts or culture media. Various methods, such as introducing the affinity tags, have been developed to promote efficient recovery and purification of recombinant proteins. These affinity tags include a variety of proteins, domains, or peptides that are genetically engineered at the N- or C- terminus of expressed proteins. A major group of affinity tags consists of a peptide or protein that binds selectively to the transition metals immobilized on a solid support. The introduction of these affinity tags allows the recombinant proteins to be purified using immobilized metal ion affinity chromatography (IMAC). Since being introduced in 1975 by Porath et al. (Porath, J., et al., 1975), IMAC has been proven to be a powerful affinity method which takes advantage of the interaction between biomolecules and transition metals.

Histidine-tags are the most widely used affinity tags. Histidine-tagged proteins have a high selective affinity for Ni2+ and several other metal ions. Purification of the histidine-tagged protein using IMAC occurs on the basis of selective interactions between histidine-tags and the metal ions within an immobilized metal chelate (Hochuli, E., et al., 1988; Porath, J., 1992; Hage, D.S., 1999). In other words, the histidine-tagged protein is the strongest binder among all the proteins in a crude sample extract. Moreover, the use of histidine tags has several merits: histidine tags are small (only six amino acids) and therefore structurally and functionally benign; histidine tags can be engineered at either the N- or C- terminus of expressed proteins, which allows some control of immobilization orientation; the interaction between histidine tags and Ni2+ with high binding stability is reversible, immobilized proteins can be eluted by lowering the pH or competitively eluted with imidazole.

Histidine-tagged protein expression in E. coli can accumulate in two main forms, as soluble proteins in biologically active forms or as inclusion bodies that are devoid of biological activity. Although alteration of expression conditions can sometimes avoid inclusion bodies, the best available tools to date have been fusion tags that increase the solubility of expressed

Affinity Chromatography

358

proteins. Various large affinity tags, such as glutathione-S-transferase (GST), maltose-binding protein (MBP), N-utilization substance A (NusA) and thioredoxin (Trx), have been used to promote the solubility of the recombinant protein. Cleavage of these affinity tags often re-introduces problems with solubility and stability.

Moloney murine leukemia virus reverse transcriptase (MMLV RT) is one of the most widely used enzyme in reverse transcription polymerase chain reaction (RT-PCR) and RNA specific amplification due to its high catalytic activity and fidelity (Kimmel & Berger, 1987; Kievits et al., 1991). In the process of prokaryotic expression, MMLV RT tends to be poorly soluble and prone to molecular aggregation. These aggregated proteins are in general misfolded and thus biologically inactive. The limited solubility of MMLV RT has been a major difficulty in mass production of the enzyme in prokaryotic system.

In this work, we presented an easy protocol established for the soluble expression and purification of recombinant MMLV RT. Six-histidine tag was used in the purification of the recombinant MMLV RT through metal ion affinity chromatography. To improve the solubility of expressed enzyme, five hydrophobic residues within the connection domain of an N-terminally truncated MMLV RT were substituted with Lysine residue in our study. The substitution significantly improved the solubility of MMLV RT and had no impact on the reverse transcriptase activity. We expect that the approach used in this study will be available to improve solubility for other problems.

2. Materials

E. coli strain DH5α and pET15b expression vector was purchased from Promega Company. Bacterial strain BL21 (DE3) plsS was kindly gifted by professor Bing-ren Huang from Peking Union Medical College. PMD18-T vector, pfu DNA polymerase, universal Taq polymerase, and restriction endonuclease Nde I/Xho I were purchased from TaKaRa Company. Protein purification was carried out using Ni-NTA resin (Beijing Dingguo Company). Shanghai Shenggong Company provides both primer synthesis and DNA sequencing. DNA gel extraction Kit, PCR product purification kit and plasmid DNA extraction kit were purchased from Beijing Tiangen Biotech Company.

3. Methods

3.1 Construction of the recombinant plasmid pET15b-MMLV-RT

3.1.1 Preparation of the MMLV RT coding sequence

To obtain the full-length MMLV RT gene, primers (sense primer RT-F: 5′-ATC TAG CTA CAT ATG ACA TGG CTG TCT GAT TTT-3′ and antisense primer RT-R: 3′-GAT TTT CTC CCA GTC TCT ATT GAG CTC ATA GCC -5′) with restriction enzyme site Nde I and Xho I were designed according to the sequence of MMLV RT gene reported by Telesnitsky A et al. In order to avoid the incorporation of base substitutions, PCR was performed with pfu DNA polymerase. The PCR product was then purified and T-A cloned into pMD18-T vector. The presence of MMLV RT gene in the recombinant plasmid was confirmed by 1% agarose gel electrophoresis. As shown in Figure 1, the expected band of about 1800 bp on agarose gel electrophoresis was detected.

Soluble Expression and Purification of Histidine-Tagged | Moloney Murine Leukemia Virus Reverse Transcriptase by Ni-NTA Affinity Chromatography

359

Fig. 1. The amplification of MMLV RT gene PCR products of MMLV RT; M. DNA Marker

3.2 Site-directed mutagenesis of MMLV RT gene

The point mutation L432K, V433K, I434K, L435K and A436K were made by PCR based site-directed mutagenesis. The primers used to make these mutations were as follows:

PF-L432K: 5′-G GGA CAG CCG AAA GTA ATA TTG GCC C -3′

PR-L432K: 5′-G GGC CAA TAT TAC TTT CGG CTG TCC C -3′

PF-V433K: 5′-G GGA CAG CCA CTT AAG ATT CTG GCC C-3′

PR-V433K: 5′-G GGC CAG AAT CTT AAG TGG CTG TCC C-3′

PF-I434K: 5′-CAG CCA CTA GTC AAG CTT GCC CCC CAT G-3′

PR-I434K: 5′-CAT GGG GGG CAA GCT TGA CTA GTG GCT G-3′

PF-L435K: 5′-CA CTA GTC ATT AAG GCC CCC CAT GCA G-3′

PR-L435K: 5′-CTG CAT GGG GGG CCT TAA TGA CTA GTG-3′

PF-A436K: 5′-CTA GTC ATT CTG AAG CCC CAT GCA GTA G-3′

PR-A436K: 5′-CTA CTG CAT GGG GCT TCA GAA TGA CTA G-3′

The underlined bases represent the mutated codon for lysine. PCR based site-directed mutagenesis was composed of two rounds of PCR. The detailed mechanism of the site-directed mutagenesis was shown as Figure 2.


360

Fig. 2. The mechanism of the site-directed mutagenesis

The first round of PCR used two pairs of primers RT-F and PR (including PR-L432K, PR-V433K, PR- I434K, PR- L435K, PR- A436K) and PF (including PF-L432K, PF-V433K, PF- I434K, PF- L435K, PF- A436K) and RT-R to create two products with the mutated sequence. The two first rounds of PCR produced two overlapping fragments of the original template, both containing the mutation within the overlap region. These two PCR products are annealed and then subjected to a second round of PCR with primers RT-F and RT-R to generate the entire fragment with the mutation. The final PCR product after second round of PCR was then T-A cloned into pMD18-T vector. The recombinant plasmid was named pMD18-T-MMLV-RT.

3.3 Preparation of the recombinant expression vector pET15b-MMLV-RT

To generate the expression vector pET15b-MMLV-RT, the recombinant plasmid pMD18-T-MMLV-RT was digested with Nde I and Xho I before insertion into the Nde I-Xho I digested pET15b vector. The recombinant expression vector was then confirmed by both enzyme digestion and sequence analysis (Figure 3). The construction of recombinant plasmid was named pET15b-MMLV-RT. Results showed that pET15b-MMLV-RT was successfully constructed.

4. Expression and purification of the mutant MMLV RT protein

4.1 Expression of the mutant MMLV RT protein

The recombinant plasmid pET15b-MMLV-RT was transformed into competent E. coli BL21 (DE3)plysS cells using standard protocols. Positive colonies were selected on LB plates


361

containing 100 μg/ml Amp. A single colony was inoculated into 1ml of LB+Amp (100 μg/ml) and incubated with shaking (220 rpm) overnight at 37°C before transferring into 9 ml LB medium. Continue incubating the culture to an OD550 of 0.6, the expression of mutant MMLV RT was induced by the addition of IPTG to the culture at a final concentration of 1mmol/L. The incubation continued for 3.5h.

1. pET15b; 2. pET15b-MMLV-RT digested by Nde I/Xho I and yielded two fragments:5708bp and 1782bp; 3. PCR products of MMLV RT (1782bp); M. DNA Marker

Fig. 3. Identification results of the recombinant plasmid pET15b-MMLV-RT by two restriction enzymes digestion.

After incubation, the cells were harvested by centrifugation at 5000g for 5min at 4°C. Resuspend the cells in 0.25 ml volume of cold 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, and centrifuge again as above. Store the cells at -70°C. The expression of MMLV RT was assessed by analysis of total cell protein on an SDS-PAGE followed by Coomassie staining. As shown in Figure 4, the MMLV RT fusion protein with 6 His on the N-terminus was obtained according to the SDS-PAGE analysis which showed a clear band of about 66.2 kDa, while no band was found in the control group without IPTG induction.

C1-C4: Recombinant plasmid pET15b-MMLV-RT without IPTG; I1-I4: Recombinant plasmid pET15b-MMLV-RT after IPTG induction. Mp: Protein marker;

Fig. 4. Expressions of MMLV RT/6His recombinant protein


362

In order to determine the solubility of the fusion protein expressed by the selected colony, the IPTG induced E. coli was collected and disrupted. Protein expressed in the supernatant and precipitate was detected by SDS-PAGE. As shown in Figure 5, SDS-PAGE analysis showed that the fusion protein was mainly expressed in the supernatant. This indicated that the introduction of the point mutations improved significantly in the solubility of the MMLV RT enzyme.

1: Recombinant plasmid pET15b-MMLV-RT without IPTG; 2: Precipitate; 3: Supernatant of bacterial body after ultrasonic disruption; Mp: Protein marker

Fig. 5. Identification of solubility properties of MMLV RT/6His

4.2 Purification of the mutant MMLV RT protein

The expressed mutant MMLV RT was then purified using the histidine-tagged protein purification method. The procedure of the histidine-tagged protein purification method is shown as Figure 6. Histidine-tagged proteins have very high affinity for Ni2+ and a variety of other immobilized metal ions, such as Co2+, Cu2+ and Zn2+ ( Porath, J. & Olin, 1983; Porath, J., 1988; Sulkowski, E., 1989). Consequently, histidine-tagged protein is selectively bound to metal-ion-charged media while other cellular proteins bind weakly or are washed out during the washing step before elution. Imidazole is the most common elution agent. Low concentration of imidazole in the binding buffer can reduce nonspecific binding of host cell proteins. While high concentration of imidazole (at least 200mM) with low pH (e.g., 0.1M glycine-HCl, pH 2.5) or an excess of strong chelator (e.g., EDTA) is required in the process of elution and recovery of captured histidine-tagged protein from the IMAC column.

For the purification of the recombinant protein, 3 ml of the supernatant was collected and purified by Ni-NTA affinity columns according to the manufacture’s instructions. Non-specific binding proteins were washed with Buffer NTA (20mM Tris-HCl pH 7.9, 0.5M NaCl, 10% Glycerol). MMLV RT was eluted with Buffer NTA containing different concentration of imidazole (20mM, 60mM, 100mM, 200mM and 300mM). SDS-PAGE analysis indicated that the concentration of the protein in the elution increases with increasing concentration of imidazole. As shown in Figure 7, 300mM imidazole in the


363

elution buffer was the optimal condition and was used in the present study. The storage buffer (5×) (250 mmol/L Tris-HCl (pH 8.3), 375 mmol/L KCl, 15 mmol/L MgCl2 and 50 mmol/L DTT) was used in the desalination of the purified protein.

Fig. 6. The procedure of the histidine-tagged protein purification method

1-6: MMLV-RT/6His eluted by Buffer NTA with different concentration of imidazole ( 300mM, 200mM, 100mM, 60mM, 20mM and 0mM) Mp: Protein marker

Fig. 7. The result of MMLV-RT/6His recombinant protein after elution and purification


364

5. Determination of the MMLV RT enzyme activity

To determine the reverse transcriptase activity of the purified MMLV RT protein, the coat protein gene of RNA bacteriophage MS2 was amplified by RT-PCR using MS2 genomic RNA as the template with the following primers: sense primer: 5'-CCT TTC GGG GTC CTG CTC AAC TT-3' and antisense primer: 5'-CTT AAG CTT CTT CGA CAT GGGTAA TCC T-3'.

Reverse transcription was performed in a 10 μl reaction volume containing 0.5 μl sense/antisense primer (10μM), 2 μl 5 × RT Buffer, 0.5μl MMLV RT, 0.25 μl RNase inhibitor and ddH2O treated with DEPC at 42°C for 15 min and ended with denaturation at 95°C for 2 min. The first-strand-cDNA synthesis was followed by PCR in a 20 μl reaction system including 2 μl cDNA, 0.5 μl sense/antisense primer (10 μM), 10 μl 2×Taq Mix and ddH2O. The conditions was as follows: 95°C for 2 min, followed by 30 cycles of 95°C for 30s, 55°C for 30s, 72°C for 2 min, and ended with a final extension step at 72°C for 7 min. RT-PCR products were then analyzed by 1% agarose gel electrophoresis. As shown in Figure 8, the clear band of about 1700bp was particular to the coat protein gene from RNA bacteriophage MS2, which indicated that the expressed MMLV RT enzyme was biological active.

1-5: Results of RT-PCR with MMLV-RT/6His; 6: Non-template control; M: DNA marker.

Fig. 8. Activity assays of MMLV-RT/6His

6. Discussion

MMLV RT is widely used in a number of routine applications, including cDNA cloning, RT-PCR, microarray analysis RACE (Rapid amplification of cDNA ends) and TMA (Transcription mediated amplification). MMLV RT is an RNA-dependent DNA polymerase that uses single-stranded RNA, DNA, or an RNA-DNA hybrid to synthesize a cDNA strand. In addition, the enzyme also contains an RNase H activity and lacks a 3→5 exonuclease activity (Aggarwal, 1990; Altschul et al., 1997). Although recombinant MMLV-RT has been produced in Escherichia coli (Tanese et al., 1985; Roth et al., 1985; Kotewicz et al., 1985), the expression and purification conditions were not detailed, and the tedious purification procedure makes it necessary to design a simple and quick method for producing soluble enzyme with biological activity.


365

One of the major difficulties in mass production of MMLV RT in prokaryotic system has proven to be the limited solubility of the enzyme. Structural analysis revealed that MMLV RT is a 75 kDa monomer comprising multiple functional domains: the fingers, palm, thumb, connection, and RNase H domains (Georgiadis et al., 1995; Das & Georgiadis, 2004; Lim et al., 2006; Cote & Roth, 2008). It has also been found that MMLV RT has a right-hand structure similar to HIV-1 RT. The finger and palm domains of MMLV RT resemble those of HIV-1 RT except that there are additional 16 residues at the N-terminal (Motakis & Parniak, 2002). For both RTs, the active site of the DNA polymerase reaction lies in the junction of the fingers and palm domains, which has three highly conserved Asp residues required for polymerase activity (Motakis & Parniak, 2002). Although functionally similar to HIV-1 RT, the MMLV RT differs both in solubility properties and detailed architecture. The relative positions of the thumb, connection and RNase H domains of MMLV RT are distinctly different from those of the p66 subunit of HIV RT (Huang et al., 1998). As shown in Figure 9, in contrast to the p66 subunit of HIV-1 RT, the MMLV RT is a far more clamp-like shaped molecule. A rotation of 18º is required to superimpose the thumb domain and connection domain of MMLV RT with the corresponding domains of the p66 subunit of HIV-1 RT. Similarly, the connection domain of MMLV RT also superimpose poorly with that of the p66 subunit of HIV-1 RT (Das & Georgiadis, 2004).

Fig. 9. Structural comparison of the MMLV RT with the p66 subunit of HIV-1 RT

The structure of MMLV RT is shown in color and the p66 subunit of HIV-1 RT is shown in gray

Here, we reported a novel and simple method to produce MMLV-RT enzyme in a soluble and active form. Five hydrophobic residues within the connection domain of MMLV RT were substituted with Lysine residue in our study. Previous structure-based sequence alignment reported that L435K is structurally equivalent to K390 in HIV-1 RT (Das &


366

Georgiadis, 2004). The introduction of the mutation in this study was found to improve the solubility of MMLV RT, which allowed purifying the N-terminal His-tagged MMLV-RT enzyme by one step Ni-NTA chromatography.

Site-directed mutagenesis has been extensively performed for a number of enzymes. A compromise between activity and stability is generally recognized in various enzymes. At times, the introduction of the mutation increase enzyme activity at the expense of the protein stability, while the increase in protein stability can also decrease enzyme activity (Yutani et al., 1987; Shoichet et al., 1995; Arnorsdottir et al., 2007; Yasukawa et al., 2010; Kusano et al., 2010). In our study, the site-directed mutagenesis resulted an increase in solubility of MMLV RT and did not affect the enzyme activity.

For the purification of the fusion protein, histidine-tag was engineered at N- terminus of MMLV RT in our research. After the purification of MMLV RT, there is no need to remove the histidine-tag, as it is small and generally less disruptive than other tags to the properties of the expressed proteins. Our results demonstrated the feasibility of the histidine-tag approach in the purification of recombinant MMLV RT.

Conclusively, we devised a novel and simple method to express and purify soluble MMLV-RT with biological activity. This method may lend itself to the production of other recombinant enzymes, which usually form inclusion body when expressed in prokaryotic system.

7. Reference

Aggarwal, A.K. (1990). Crystallization of DNA Binding Proteins with Oligodeoxynucleotides. Methods, Vol.1, No.1, (August 1990), pp. 83-90, ISSN 1046-2023

Altschul, S.F.; Madden, T.L.; Schaffer, A.A.; Zhang, J.H.; Zhang, Z.; Miller, W. & Lipman, D.J. (1997). Gapped BLAST and PSI-BLAST: a New Generation of Protein Database Search Programs. Nucleic Acids Research. Vol.25, No.17, (July 1997), pp. 3389-3402, ISSN 0305-1048

Arnorsdottir, J.; Helgadottir, S.; Thorbjarnardottir, S.H.; Eggertsson, G. & Kristjansson, M.M. (2007). Effect of Selected Ser/Ala and Xaa/Pro Mutations on the Stability and Catalytic Property of a Cold Adapted Subtilisin-like Serine Proteinase. Biochimica et Biophysica Acta-Proteins & Proteomics. Vol.1774, No.6, (June 2007), pp. 749-755, ISSN 1570-9639

Cote, M.L. & Roth, M.J. (2008). Murine Leukemia Virus Reverse Transcriptase: Structural Comparison with HIV-1 Reverse Transcriptase. Virus Research, Vol.134, No.1-2, (June 2008), pp. 186-202, ISSN 0168-1702

Das, D. & Georgiadis, M.M. (2004). The Crystal Structure of the Monomeric Reverse Transcriptase from Moloney Murine Leukemia Virus. Structure. Vol.12, No.5, (May 2004), pp. 819-829, ISSN 0969-2126

Georgiadis, M.M.; Jessen, S.M.; Ogata, C.M.; Telesnitsky, A.; Goff, S.P. & Hendrickson W.A. (1995). Mechanistic Implications from the Structure of a Catalytic Fragment of Moloney Murine Leukemia Virus Reverse Transcriptase. Structure, Vol.3, No.9, (September 1995), pp. 879-892, ISSN 0969-2126


367

Hage, D.S. (1999). Affinity Chromatography: A Review of Clinical Applications. Clinical Chemistry, Vol.45, (1999), pp. 593-615, ISSN 0009-9147

Hochuli, E.; Bannwarth, W.; Dobeli, H.; Gentz, R. & Stuber, D. (1988). Genetic Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chlelate Adsorbent. Nature Biotechnology, Vol.6, (1988), pp. 1321-1325, ISSN 1087-0156

Huang, H.; Chopra, R.; Verdine, G.L. & Harrison, S.C. (1998). Structure of a Covalently Trapped Catalytic Complex of HIV-1 Reverse Transcriptase: Implications for Drug Resistance. Science, Vol.282, No.5394, (November 1998), pp. 1669-1675, ISSN 0036-8075

Kievits, T.; van Gemen, B.; van Strijp, D.; Schukkink, R.; Dircks, M.; Adriaanse, H.; Malek, L.; Sooknanan, R. & Lens, P. (1991). NASBATM Isothermal Enzymatic in Vitro Nucleic Acid Amplification Optimized for the Diagnosis of HIV-1 Infection. Journal of Virological Methods, Vol.35, No.3, (December 1991), pp. 273-286, ISSN 0166-0934

Kimmel, A.R. & Berger, S.L. (1987). Preparation of cDNA and the Generation of cDNA Libraries: Overview. Methods in Enzymology, Vol.152, (1987), pp. 307-316, ISSN 0076-6879

Kotewicz, M.L.; D’Alessio, J.M.; Driftmier, K.M.; Blodgett, K.P. & Gerard, G.F. (1985). Cloning and Overexpression of Moloney Murine Leukemia Virus Reverse Transcriptase in Escherichia coli. Gene, Vol.35, No.3, (1985), pp. 249-258, ISSN 0378-1119

Kusano, M.; Yasukawa, K. & Inouye, K. (2010). Effects of the Mutational Combinations on the Activity and Stability of Thermolysin. Journal of Biotechnology, Vol.147, No.1, (May 2010), pp. 7-16, ISSN 0168-1656

Lim, D.; Gregorio, G.G.; Bingman, C.; Martinez-Hackert, E.; Hendrickson, W.A.; & Goff, S.P. (2006). Crystal Structure of the Moloney Murine Leukemia Virus RNase H Domain. Journal of Virology, Vol.80, No.17, (September 2006), pp. 8379-8389, ISSN 0022-538X

Motakis, D. & Parniak, M.A. (2002). A Tight-binding Mode of Inhibition is Essential for Anti-human Immunodeficiency Virus Type I Virucidal Activity of Nonnucleoside Reverse Transcriptase Inhibitors. Antimicrobial Agents and Chemotherapy, Vol.46, No.6, (June 2002), pp. 1851-1856, ISSN 0066-4804

Porath, J.; Carlsson, J.; Olsson, J. & Belfrage, G. (1975). Metal Chelate Affinity Chromatography, A New Approach to Protein Fractionation. Nature, Vol.258, No.5536, (1975), pp. 598-599, ISSN 0028-0836

Porath, J. and Olin, B. (1983). Immobilized Metal Affinity Adsorption and Immobilized Metal Affinity Chromatography of Biomaterials. Serum Protein Affinities for Gel-immobilized Iron and Nickel Ions. Biochemistry, Vol.22, No.7, (March 1993), pp. 1621-1630, ISSN 0006-2960

Porath, J. (1988). IMAC-Immobilized Metal Ion Affinity Based Chromatography, Trac-Trends in Analytical Chemistry, Vol.7, No.7, (August 1988), pp. 254-259, ISSN 0165-9936

Porath, J. (1992). Immobilized Metal Ion Affinity Chromatography. Protein Expression and Purification, Vol.3, No.4, (August 1992), pp. 263-281, ISSN 1046-5928

Roth, M.J.; Tanese, N. & Goff, S.P. (1985). Purification and Characterization of Murine Retroviral Reverse Transcriptase Expressed in Escherichia coli. Journal of Biological Chemistry, Vol.260, No.16, (August 1985), pp. 9326-9335, ISSN 0021-9258


368

Shoichet, B.K.; Baase, W.A.; Kuroki, R. & Matthews, B.W. (1995). A Relationship Between Protein Stability and Protein Function. Proceedings of the National Academy of Sciences of the United States of America, Vol.92, No.2, (January 1995), pp. 452-456, ISSN 0027-8424

Sulkowski, E. (1989). The Saga of IMAC and MIT. Bioessays. Vol.10, No.5, (May 1989), pp. 170-175, ISSN 0265-0247

Tanese, N.; Roth, M. & Goff, S.P. (1985). Expression of Enzymatically Active Reverse Transcriptase in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, Vol.82, No.15, (August 1985), pp. 4944-4948, ISSN 0027-8424

Yasukawa, K.; Agata, N. & Inouye, K. (2010). Detection of cesA mRNA from Bacillus Cereus by RNA-specific Amplification. Enzyme and Microbial Technology, Vol.46, No.5, (April 2010), pp. 391-396, ISSN 0141-0229

Yutani, K.; Ogasahara, K.; Tsujita, T. & Sugino, Y. (1987). Dependence of Conformational Stability on Hydrophobicity of the Amino Acid Residue in a Series of Variant Proteins Substituted at a Unique Position of Tryptophan Synthase α Subunit. Proceedings of the National Academy of Sciences of the United States of America, Vol.84, No.13, (July 1987), pp. 4441-4444, ISSN 0027-8424

Soluble Expression and Purification of Histidine-Tagged ...€¦ · While high concentration of...

Documents

Transcript of Soluble Expression and Purification of Histidine-Tagged ...€¦ · While high concentration of...