title: Versatile vector suite for the extracytoplasmic production and purification of heterologous His-tagged proteins in Lactococcus lactis.
abstract: Recent studies have shown that the Gram-positive bacterium Lactococcus lactis can be exploited for the expression of heterologous proteins; however, a versatile set of vectors suitable for inducible extracellular protein production and subsequent purification of the expressed proteins by immobilized metal affinity chromatography was so far lacking. Here we describe three novel vectors that, respectively, facilitate the nisin-inducible production of N- or C-terminally hexa-histidine (His6)-tagged proteins in L. lactis. One of these vectors also encodes a tobacco etch virus (TEV) protease cleavage site allowing removal of the N-terminal His6-tag from expressed proteins. Successful application of the developed vectors for protein expression, purification and/or functional studies is exemplified with six different cell wall-bound or secreted proteins from Staphylococcus aureus. The results show that secretory production of S. aureus proteins is affected by the position, N- or C-terminal, of the His6-tag. This seems to be due to an influence of the His6-tag on protein stability. Intriguingly, the S. aureus IsdB protein, which is phosphorylated in S. aureus, was also found to be phosphorylated when heterologously produced in L. lactis, albeit not on the same Tyr residue. This implies that this particular post-translational protein modification is to some extent conserved in S. aureus and L. lactis. Altogether, we are confident that the present vector set combined with the L. lactis expression host has the potential to become a very useful tool in optimization of the expression, purification and functional analysis of extracytoplasmic bacterial proteins.
authors.affiliation: Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, P.O. Box 30001, 9700 RB, Groningen, The Netherlands.
authors.forename: Jolanda
authors.initials: J
authors.lastname: Neef
authors.affiliation: Department of Medical Microbiology, University Medical Center Utrecht, PO G04.614, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.
authors.forename: Fin J
authors.initials: FJ
authors.lastname: Milder
authors.affiliation: Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, P.O. Box 30001, 9700 RB, Groningen, The Netherlands.
authors.forename: Danny G A M
authors.initials: DG
authors.lastname: Koedijk
authors.affiliation: Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, P.O. Box 30001, 9700 RB, Groningen, The Netherlands.
authors.forename: Marindy
authors.initials: M
authors.lastname: Klaassens
authors.affiliation: Department of Medical Microbiology, University Medical Center Utrecht, PO G04.614, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.
authors.forename: Erik C
authors.initials: EC
authors.lastname: Heezius
authors.affiliation: Department of Medical Microbiology, University Medical Center Utrecht, PO G04.614, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.
authors.forename: Jos A G
authors.initials: JA
authors.lastname: van Strijp
authors.affiliation: Institut für Mikrobiologie, Ernst-Moritz-Arndt Universität Greifswald, Friedrich-Ludwig-Jahn-Str. 15, D-17489, Greifswald, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Otto
authors.affiliation: Institut für Mikrobiologie, Ernst-Moritz-Arndt Universität Greifswald, Friedrich-Ludwig-Jahn-Str. 15, D-17489, Greifswald, Germany.
authors.forename: Dörte
authors.initials: D
authors.lastname: Becher
authors.affiliation: Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, P.O. Box 30001, 9700 RB, Groningen, The Netherlands.
authors.forename: Jan Maarten
authors.initials: JM
authors.lastname: van Dijl
authors.affiliation: Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, P.O. Box 30001, 9700 RB, Groningen, The Netherlands. g.buist@umcg.nl.
authors.forename: Girbe
authors.identifier: http://orcid.org/0000-0002-1583-3713
authors.initials: G
authors.lastname: Buist
mesh_terms: D002846:Chromatography, Affinity; D005822:Genetic Vectors; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism*; D009561:Nisin / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000235:genetics; D015533:Transcriptional Activation / Q000187:drug effects
chemical_list: D011993:Recombinant Fusion Proteins; D009561:Nisin; D010447:Peptide Hydrolases
keywords: Expression vectors; Histidine tag; L. lactis; NICE; Secretion; Usp45