Publication

A Lactococcus lactis expression vector set with multiple affinity tags to facilitate isolation and direct labeling of heterologous secreted proteins

Pastrana, F. R., Neef, J., van Dijl, J. M. & Buist, G., Nov-2017, In : Applied Microbiology and Biotechnology. 101, 22, p. 8139-8149 11 p.

Research output: Contribution to journalArticleAcademicpeer-review

The gram-positive bacterium Lactococcus lactis is a useful host for extracellular protein production. A main advantage of L. lactis over other bacterial expression systems is that lactococcal cells display low levels of autolysis and proteolysis. Previously, we developed a set of vectors for nisin-inducible extracellular production of N- or C-terminally hexa-histidine (His(6))-tagged proteins. The present study was aimed at expanding our portfolio of L. lactis expression vectors for protein purification and site-specific labeling. Specifically, we present two new groups of vectors allowing N- or C-terminal provision of proteins with a Strep-tag II or AVI-tag. Vectors for AVI-tagging encode an additional His(6)-tag for protein purification. Another set of vectors allows removal of N-terminal Strep- or His(6)-tags from expressed proteins with the tobacco etch virus protease. Two possible applications of the developed vectors are presented. First, we show that Strep-tagged LytM of Staphylococcus aureus in the growth medium of L. lactis can be directly bound to microtiter plates coated with an affinity reagent and used for enzyme-linked immunosorbent assays. Second, we show that the AVI-tagged Sle1 protein from S. aureus produced in L. lactis can be directly biotinylated and fluorescently labeled. The fluorescently labeled Sle1 was successfully applied for S. aureus re-binding studies, allowing subcellular localization by fluorescence microscopy. In conclusion, we have developed a set of expression vectors that enhances the versatility of L. lactis as a system for production of proteins with tags that can be used for affinity purification and site-specific protein labeling.

Original languageEnglish
Pages (from-to)8139-8149
Number of pages11
JournalApplied Microbiology and Biotechnology
Volume101
Issue number22
Publication statusPublished - Nov-2017

    Keywords

  • Lactococcus lactis, Expression vector, Strep-tag, AVI-tag, Staphylococcus aureus, CONTROLLED GENE-EXPRESSION, STAPHYLOCOCCUS-AUREUS, PURIFICATION, PEPTIDOGLYCAN, RECOGNITION, SYSTEM, WALL, ANTIGENS, BACTERIA, DOMAINS

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