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

• Published in: Applied microbiology and biotechnology Vol. 101; no. 22; pp. 8139 - 8149
• Main Authors: Romero Pastrana, Francisco, Neef, Jolanda, van Dijl, Jan Maarten, Buist, Girbe
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.11.2017; Springer; Springer Nature B.V
• Subjects: Affinity; Applied Genetics and Molecular Biotechnology; Autolysis; Bacteria; Bacterial proteins; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Biomedical and Life Sciences; Biotechnology; Chromatography, Affinity; Enzyme-Linked Immunosorbent Assay; Expression vectors; Fluorescence; Fluorescence microscopy; Genetic Vectors; Gram-positive bacteria; Histidine; Immunoassays; Labeling; Laboratory equipment; Lactococcus; Lactococcus lactis; Lactococcus lactis - genetics; Lactococcus lactis - metabolism; Life Sciences; Localization; Microbial Genetics and Genomics; Microbiology; Nisin; Oligopeptides - chemistry; Physiological aspects; Protein expression; Protein purification; Proteins; Proteolysis; Purification; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Staphylococcus aureus; Staphylococcus aureus - genetics; Staphylococcus aureus - metabolism; Tags; Tobacco; Viruses; Strep-tag; AVI-tag; Expression vector; Lactococcus lactis; Staphylococcus aureus
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-017-8524-x

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.