Phuc H. B. Nguyen, Hieu H. Nguyen, Gwen R. Jordaan, Roger A. Acey

Department of Chemistry and Biochemistry, California State University 1250 N. Bellflower Blvd. Long Beach CA 90840 USA

Abstract:We have been developing a method for improving the expression and yield of Artemia

metallothionein (MT). The protein has the unique ability to selectively bind heavy metals, e.g., lead,

mercury, gold, and uranium. Therefore, using MT, we are developing a “heavy metal sponge” for the

removal and/or recovery of toxic and precious metal from aqueous solutions. Our goal here was to

develop a fermentation process to enhance the yield and simplify the purification of MT. This

involved generating an MT N-his-tagged SUMO (small ubiquitin-like modifier) chimeric protein. The

chimeric MT was cloned and the recombinant protein expressed using an E. Coli inducible T7

expression system. The SUMO tag was incorporated to enhance the expression and solubility of MT.

The media used for fermentation was supplemented with a trace metal solution and ammonium

chloride, and fermentation was maintained at pH 7.0. There was an 8-fold increase in cell mass

relative to expression in flasks. Purification was by affinity chromatography using Ni2+-NTA resin. The

chimeric protein retained its metal binding activity. The concentration of eluted protein was four

times more than previously purified by other methods. Analysis of the eluted protein by SDS-PAGE

showed a 22 kDa protein consistent with the size of the MT fusion protein. Supported in part by a

grants from CSUPERB and the CSULB Mini-Grant Program and private donations. Covered by US

Patent number7,273,962

Conclusions:

• N-His-SUMO-MT has been successfully expressed and purified.

• Fermentation increased cell yield 8 fold compared to shake-flask methods

• N-His-SUMO-MT significantly simplifies the purification of the protein and increases yield

relative to former purification procedures.

Results:

Acknowledgements: CSUPERB & CSULB Mini Grant Program

Introduction:

Metallothionien (MT) is a low molecular weight, cysteine rich metal binding protein devoid of aromatic amino acids. MT has the unique ability to selectively bind toxic (or precious) heavy metals, e.g., lead, mercury, and gold. It does not bind biologically essential metals such as sodium and calcium. The metal binding activity is active under extremes of pH (4 to 10) and temperature (4 to 100 oC). Metal binding also occurs instantaneously. The dissociation constants (Kd) for the metal-protein interaction are in the range of 10 -12 to 10 -15. These properties make MT are well suited for environmental applications requiring toxic or precious metal removal and/or recovery. We are working to improve the level of expression of MT using a fermentation protocol. To simplify the purification method, an N-his-tagged SUMO MT fusion construct was made. The protein then was expressed in an inducible T7 expression system in E. coli.

Enhanced Expression and Purification of a SUMO-Metallothionein

Chimeric Protein Using an Improved Fermentation Method

Materials and Methods:Cloning of N-his-tagged SUMO MT fusion construct

The forward primer incorporated the 5’ end of the MT coding sequence and an 18 bp overlap

corresponding to the C-terminus of the SUMO protein. The reverse primer included the 3’ end of the

MT coding sequence and an 18 bp overlap containing a stop codon and vector sequence. PCR

amplification of 5 ng of MT coding sequence produced a 180 bp product. The PCR product was gel

purified. To produce SUMO/MT clones, ligation independent cloning was performed by incubating 25

ng of N-his-SUMO-pETite vector (Lucigen), 59 ng of purified PCR product and 40 μl HI-Control 10G

chemically competent cells (Lucigen) on ice for 30 min. The mixture was heat-shocked at 42ᵒC for 45

sec followed by 2 min on ice. Cells were incubated with 960 μl recovery medium at 37ᵒC for 1 h. Cells

were plated on YT agar containing 30 μg/ml kanamycin and 1% glucose. Clones were selected and

verified for the SUMO/MT insert by PCR screening. Plasmid DNA was purified using Qiagen midi

columns. The SUMO/MT sequence was verified by DNA sequencing. To establish SUMO/MT expression

clones, 6 ng of purified plasmid was transformed into HI-Control BL21(DE3) chemically competent cells

(Lucigen) as described previously. Confirmation of insert and SUMO/MT sequence was verified as

previously described. The construct was referred to as pCODAsumo-mt.

Expression of MT and N-his-tagged SUMO MT using shake-flask technique

The MT gene was cloned into pET 11a. The construct is referred to as pCODAmt. Recombinant MT was

expressed in BL-21 E.coli. LB Broth was inoculated with either pCODAmt or pCODAsumo-mt and incubated

overnight at 37oC and 220 rpm. The following morning, a one liter LB culture was inoculated with 14 ml

of the overnight culture. The cells were cultured to an A560 between 0.4-0.6. IPTG was added to the

culture to a final concentration of 1mM to induce expression of MT.

Expression and purification of N-his-tagged SUMO MT using fermentation technique

N-his-tagged SUMO MT was expressed in BL-21 E.coli. LB broth was inoculated with pCODAsumo-mt and

incubated overnight at 37oC and 220 rpm. Next morning, 2.5 liter of fermentation media was

inoculated with 125 ml of overnight culture. The culture was maintained at 37oC and pH=7.00. After

the nutrition was exhausted, 25 g of glucose and 0.5 g of ammonium chloride was added. The cells

were cultured to an A660 of 15. IPTG was added to the culture to a final concentration of 1mM to

induce expression of MT. Temperature was lowered to 20oC for 3 hours induction period.

Purification of N-his-tagged SUMO MT using Affinity Chromatography

Cells were collected and lysed by sonication in 10mM Tris pH=8.0, 1ppm Zn2+, 0.1mM DTT, and 0.5mM

PMSF. The lysate was placed in boiling water for 10 minutes then clarified by centrifugation. Five to ten

milligrams protein per milliliter Ni-NTA agarose was mixed. The mixture was incubated in 50 mM

Na2PO4, 300 mM NaCl overnight at 4oC. N-his-tagged SUMO MT was eluted with 400 mM imidazole.

Protein concentration was determined and the purity of the preparation determined by SDS-PAGE 4-

12% Bis-Tris gel.

Figure 3. Cell Mass per Liter Culture Collected from Shaker Flasks or the Fermentor using pCODASUMO-MT . With the shaker flasks, cells were cultured to an A560 between 0.4-0.6. IPTG was added to the culture to a final concentration of 1mM to induce expression of MT. In fermentation method, the cells were induced at an A660 of 15.

References: Acey, R.A., Harpham, B., and Mustillo, M. Metal Binding Proteins and Associated Methods, US Patent # 7,135,605. Issued 11/14/06.Acey, R.A., Compositions and Methods for Removing Heavy Metals from Contaminated Samples

Using Membranes Provided with Purified Artemia Metallothionein(MT) Peptides, US Patent #7,237,962. Issued 9/25/07

Figure 1. N-His-SUMO-MT DNA Sequence and pETite Fusion Plasmid. The forward primer incorporated the 5’ end of the MT coding sequence and an overlap corresponding to the C-terminus of the SUMO protein. The reverse primer included the 3’ end of the MT coding sequence and an overlap containing a stop codon and vector sequence. PCR amplification of MT coding sequence was produced and gel purified. Ligation independent cloning was done to produce SUMO/MT clones.

Figure 2. Growth Curve of E.coli containing pCODASUMO-MT in the First 5 Hours Using Fermentation and Shaker Flasks. Cells were inoculated overnight in LB Broth. The following morning, the overnight culture was transferred to either 1 L culture in shake flasks or 2.5 L medium in a fermentor.

Figure 4. SDS-PAGE Analysis of Purified N-His-SUMO-MT by Affinity Chromatorgahy. The prominent band at 22KDa was identified to be N-His-SUMO-MT. Cell lysate was mixed with Ni-NTA agarose. The mixture was incubated overnight at 4oC. N-his-tagged-SUMO-MT was eluted with 400 mMimidazole. Sample from each elution fraction was loaded onto SDS-PAGE 4-12% Bis-Tris gel.

Figure 5. Protein Yield of MT Purification from E.coli containing pCODAMT

and pCODASUMO-MT Plasmids. MT from pCODAMT was purified using gel-filtration(molecular exclusion and ion exchange). N-His-SUMO-MT from pCODASUMO-MT was purified with Ni affinity chromatography.

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