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Engineering of Cysteine Residues Leads to Improved Production of a Human Dipeptidase Enzyme in E. coli

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Abstract

Low yields, poor folding efficiencies and improper disulfide bridge formation limit large-scale production of cysteine-rich proteins in Escherichia coli. Human renal dipeptidase (MDP), the only human β-lactamase known to date, is a homodimeric enzyme, which contains six cysteine residues per monomer. It hydrolyses penem and carbapenem β-lactam antibiotics and can cleave dipeptides containing amino acids in both d- and l-configurations. In this study, MDP accumulated in inactive form in high molecular weight, disulfide-linked aggregates when produced in the E. coli periplasm. Mutagenesis of Cys361 that mediates dimer formation and Cys93 that is unpaired in the native MDP led to production of soluble recombinant enzyme, with no change in activity compared with the wild-type enzyme. The removal of unpaired or structurally inessential cysteine residues in this manner may allow functional production of many multiply disulfide-linked recombinant proteins in E. coli.

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Acknowledgements

The authors wish to thank Timothy Smyth and Sylvain Robin for helpful discussions and Tewfik Soulimane for technical assistance. Barbara Leiting of Merck kindly provided cilastatin sodium. This work was funded by the Enterprise Ireland Science and Technology agency grant SC/2001/432 (ROD) and by the Higher Education Authority through the Programme for Research in Third Level Institutions (RR).

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Authors and Affiliations

  1. Department of Chemical and Environmental Sciences and Materials and Surface Science Institute, University of Limerick, National Technology Park, Limerick, Ireland

    Ronan O’Dwyer, Rafia Razzaque, Xuejun Hu & J. Gerard Wall

  2. Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, 35294-2170, USA

    Rafia Razzaque & Susan K. Hollingshead

  3. Medical School, Dalian University, Liao Ning Province, China

    Xuejun Hu

  4. Department of Microbiology, School of Natural Sciences, NUI Galway, Galway, Ireland

    J. Gerard Wall

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  1. Ronan O’Dwyer
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  2. Rafia Razzaque
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  4. Susan K. Hollingshead
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  5. J. Gerard Wall
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Correspondence to J. Gerard Wall.

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O’Dwyer, R., Razzaque, R., Hu, X. et al. Engineering of Cysteine Residues Leads to Improved Production of a Human Dipeptidase Enzyme in E. coli . Appl Biochem Biotechnol 159, 178–190 (2009). https://doi.org/10.1007/s12010-008-8379-9

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