Efficient expression of sortase A from Staphylococcus aureus in Escherichia coli and its enzymatic characterizations

Abstract

Background

Sortase A (SrtA) is a transpeptidase found in Staphylococcus aureus, which is widely used in site-specific protein modification. However, SrtA was expressed in Escherichia coli (E. coli) in rather low level (ranging from several milligrams to 76.9 mg/L at most). The present study aims to optimize fermentation conditions for improving SrtA expression in E. coli.

Results

Under the optimized media (0.48 g/L glycerol, 1.37 g/L tryptone, 0.51 g/L yeast extract, MOPS 0.5 g/L, PBS buffer 180 mL/L) and condition (30 °C for 8 h) in a 7-L fermentor, the enzyme activity and the yield of SrtA reached 2458.4 ± 115.9 U/mg DCW and 232.4 ± 21.1 mg/L, respectively, which were higher by 5.8- and 4.5-folds compared with initial conditions, respectively. The yield of SrtA also represented threefold increase than the previously reported maximal level. In addition, the enzymatic characterizations of SrtA (optimal temperature, optimal pH, the influence of metal irons, and tolerance to water-soluble organic solvents) were determined.

Conclusions

Enhanced expression of SrtA was achieved by optimization of medium and condition. This result will have potential application for production levels of SrtA on an industry scale. Moreover, the detailed enzymatic characterizations of SrtA were examined, which will provide a useful guide for its future application.

Keywords

Sortase A / Escherichia coli / Response surface methodology / 7-L fermentor / Enzymatic characterizations

References

[1] Beerli RR, Hell T, Merkel AS, Grawunder U. Sortase enzyme-mediated generation of site-specifically conjugated antibody drug conjugates with high in vitro and in vivo potency. PLoS ONE, 2015, 10: 131177.

[2] Bentley ML, Lamb EC, McCafferty DG. Mutagenesis studies of substrate recognition and catalysis in the sortase A transpeptidase from Staphylococcus aureus. J Biol Chem, 2008, 283: 14762-14771.

[3] Buchanan RL, Klawitter LA. The effect of incubation temperature, initial pH, and sodium chloride on the growth kinetics of Escherichia coli O157:H7. Food Microbiol, 1992, 9: 185-196.

[4] Chan L, Cross HF, She JK, Cavalli G, Martins HF, Neylon C. Covalent attachment of proteins to solid supports and surfaces via sortase-mediated ligation. PLoS ONE, 2007, 2: e1164.

[5] Glasgow JE, Salit ML, Cochran JR. In vivo site-specific protein tagging with diverse amines using an engineered sortase variant. J Am Chem Soc, 2016, 138: 7496-7499.

[6] Guo X, Wang Q, Swarts BM, Guo Z. Sortase-catalyzed peptide-glycosylphosphatidylinositol analogue ligation. J Am Chem Soc, 2009, 131: 9878-9879.

[7] Hirakawa H, Ishikawa S, Nagamune T. Design of Ca²⁺-independent Staphylococcus aureus sortase A mutants. Biotechnol Bioeng, 2012, 109: 2955-2961.

[8] Ilangovan U, Ton-That H, Iwahara J, Schneewind O, Clubb RT. Structure of sortase, the transpeptidase that anchors proteins to the cell wall of Staphylococcus aureus. Proc Natl Acad Sci USA, 2001, 98: 6056-6061.

[9] Kim SW, Chang IM, Oh KB. Inhibition of the bacterial surface protein anchoring transpeptidase sortase by medicinal plants. Biosci Biotechnol Biochem, 2002, 66: 2751-2754.

[10] Kruger RG, Dostal P, McCafferty DG. Development of a high-performance liquid chromatography assay and revision of kinetic parameters for the Staphylococcus aureus sortase transpeptidase SrtA. Anal Biochem, 2004, 326: 42-48.

[11] Lee C, Sun WJ, Burgess BW, Junker BH, Reddy J, Buckland BC, . Process optimization for large-scale production of TGF-alpha-PE40 in recombinant Escherichia coli: effect of medium composition and induction timing on protein expression. J Ind Microbiol Biotechnol, 1997, 18: 260-266.

[12] Lee KY, Shin DS, Yoon JM, Heonjoong K, Oh KB. Expression of sortase, a transpeptidase for cell wall sorting reaction, from Staphylococcus aureus ATCC 6538p in Escherichia coli. J Microbiol Biotechnol, 2002, 12: 530-533.

[13] Li Z, Nimtz M, Rinas U. The metabolic potential of Escherichia coli BL21 in defined and rich medium. Microb Cell Fact, 2014, 13: 45.

[14] Madej MP, Coia G, Williams CC, Caine JM, Pearce LA, Attwood R, . Engineering of an anti-epidermal growth factor receptor antibody to single chain format and labeling by sortase A-mediated protein ligation. Biotechnol Bioeng, 2012, 109: 1461-1470.

[15] Matsushita T, Sadamoto R, Ohyabu N, Nakata H, Fumoto M, Fujitani N, . Functional neoglycopeptides: synthesis and characterization of a new class of MUC1 glycoprotein models having core 2-based O-glycan and complex-type N-glycan chains. Biochemistry, 2009, 48: 11117-11133.

[16] Mazmanian SK, Liu G, Ton-That H, Schneewind O. Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science, 1999, 285: 760-763.

[17] Naik MT, Suree N, Ilangovan U, Liew CK, Thieu W, Campbell DO, . Staphylococcus aureus Sortase A transpeptidase. Calcium promotes sorting signal binding by altering the mobility and structure of an active site loop. J Biol Chem, 2006, 281: 1817-1826.

[18] Papaneophytou CP, Kontopidis G. Statistical approaches to maximize recombinant protein expression in Escherichia coli: a general review. Protein Expr Purif, 2014, 94: 22-32.

[19] Perry AM, Ton-That H, Mazmanian SK, Schneewind O. Anchoring of surface proteins to the cell wall of Staphylococcus aureus. III. Lipid II is an in vivo peptidoglycan substrate for sortase-catalyzed surface protein anchoring. J Biol Chem, 2002, 277: 16241-16248.

[20] Perry AM, Ton-That H, Mazmanian SK, Schneewind O. Anchoring of surface proteins to the cell wall of Staphylococcus aureus. III. Lipid II is an in vivo peptidoglycan substrate for sortase-catalyzed surface protein anchoring. J Biol Chem, 2002, 277: 16241-16248.

[21] Scott CJ, McDowell A, Martin SL, Lynas JF, Vandenbroeck K, Walker B. Irreversible inhibition of the bacterial cysteine protease-transpeptidase sortase (SrtA) by substrate-derived affinity labels. Biochem J, 2002, 366: 953-958.

[22] Tanaka T, Yamamoto T, Tsukiji S, Nagamune T. Site-specific protein modification on living cells catalyzed by Sortase. ChemBioChem, 2008, 9: 802-807.

[23] Ton-That H, Liu G, Mazmanian SK, Faull KF, Schneewind O. Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif. Proc Natl Acad Sci USA, 1999, 96: 12424-12429.

[24] Tseng CL, Leng CH. Influence of medium components on the expression of recombinant lipoproteins in Escherichia coli. Appl Microbiol Biotechnol, 2012, 93: 1539-1552.

[25] Vincentelli R, Romier C. Expression in Escherichia coli: becoming faster and more complex. Curr Opin Struct Biol, 2013, 23: 326-334.

[26] Voloshchuk N, Liang D, Liang JF. Sortase A mediated protein modifications and peptide conjugations. Curr Drug Disc Technol, 2015, 12: 205-213.

[27] Witte MD, Cragnolini JJ, Dougan SK, Yoder NC, Popp MW, Ploegh HL. Preparation of unnatural N-to-N and C-to-C protein fusions. Proc Natl Acad Sci USA, 2012, 109: 11993-11998.

[28] Wu Z, Guo X, Wang Q, Swarts BM, Guo Z. Sortase A-catalyzed transpeptidation of glycosylphosphatidylinositol derivatives for chemoenzymatic synthesis of GPI-anchored proteins. J Am Chem Soc, 2010, 132: 1567-1571.

[29] Wu Z, Guo X, Guo Z. Sortase A-catalyzed peptide cyclization for the synthesis of macrocyclic peptides and glycopeptides. Chem Commun, 2011, 47: 9218-9220.

[30] Wu Z, Guo X, Gao J, Guo Z. Sortase A-mediated chemoenzymatic synthesis of complex glycosylphosphatidylinositol-anchored protein. Chem Commun, 2013, 49: 11689-11691.

Funding

National Natural Science Foundation of China(21472070)

he Project for Jiangsu Scientific and Technological Innovation Team

Fund for Jiangsu Distinguished Professorship Program

the State Key Laboratory of Natural and Biomimetic Drugs(K20140216)

Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions

the 111 Project((No. 111-2-06))

the Jiangsu province "Collaborative Innovation Center for Advanced Industrial Fermentation" industry development program