caa a22 4500 605505640 CHVBK 20210128100623.0 cr unu---uuuuu 210128e20151101xx s 000 0 eng 10.1007/s00253-015-6778-8 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6778-8 Versatile vector suite for the extracytoplasmic production and purification of heterologous His-tagged proteins in Lactococcus lactis [Elektronische Daten] [Jolanda Neef, Fin Milder, Danny Koedijk, Marindy Klaassens, Erik Heezius, Jos van Strijp, Andreas Otto, Dörte Becher, Jan van Dijl, Girbe Buist] 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. The Author(s), 2015 L. lactis nationallicence Expression vectors nationallicence NICE nationallicence Usp45 nationallicence Histidine tag nationallicence Secretion nationallicence Neef Jolanda Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, P.O. Box 30001, 9700 RB, Groningen, The Netherlands aut Milder Fin Department of Medical Microbiology, University Medical Center Utrecht, Heidelberglaan 100, PO G04.614, 3584 CX, Utrecht, The Netherlands aut Koedijk Danny Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, P.O. Box 30001, 9700 RB, Groningen, The Netherlands aut Klaassens Marindy Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, P.O. Box 30001, 9700 RB, Groningen, The Netherlands aut Heezius Erik Department of Medical Microbiology, University Medical Center Utrecht, Heidelberglaan 100, PO G04.614, 3584 CX, Utrecht, The Netherlands aut van Strijp Jos Department of Medical Microbiology, University Medical Center Utrecht, Heidelberglaan 100, PO G04.614, 3584 CX, Utrecht, The Netherlands aut Otto Andreas Institut für Mikrobiologie, Ernst-Moritz-Arndt Universität Greifswald, Friedrich-Ludwig-Jahn-Str. 15, D-17489, Greifswald, Germany aut Becher Dörte Institut für Mikrobiologie, Ernst-Moritz-Arndt Universität Greifswald, Friedrich-Ludwig-Jahn-Str. 15, D-17489, Greifswald, Germany aut van Dijl Jan Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, P.O. Box 30001, 9700 RB, Groningen, The Netherlands aut Buist Girbe Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, P.O. Box 30001, 9700 RB, Groningen, The Netherlands aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/21(2015-11-01), 9037-9048 0175-7598 99:21<9037 2015 99 253 https://doi.org/10.1007/s00253-015-6778-8 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6778-8 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Neef Jolanda Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, P.O. Box 30001, 9700 RB, Groningen, The Netherlands aut NATIONALLICENCE 700 1- Milder Fin Department of Medical Microbiology, University Medical Center Utrecht, Heidelberglaan 100, PO G04.614, 3584 CX, Utrecht, The Netherlands aut NATIONALLICENCE 700 1- Koedijk Danny Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, P.O. Box 30001, 9700 RB, Groningen, The Netherlands aut NATIONALLICENCE 700 1- Klaassens Marindy Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, P.O. Box 30001, 9700 RB, Groningen, The Netherlands aut NATIONALLICENCE 700 1- Heezius Erik Department of Medical Microbiology, University Medical Center Utrecht, Heidelberglaan 100, PO G04.614, 3584 CX, Utrecht, The Netherlands aut NATIONALLICENCE 700 1- van Strijp Jos Department of Medical Microbiology, University Medical Center Utrecht, Heidelberglaan 100, PO G04.614, 3584 CX, Utrecht, The Netherlands aut NATIONALLICENCE 700 1- Otto Andreas Institut für Mikrobiologie, Ernst-Moritz-Arndt Universität Greifswald, Friedrich-Ludwig-Jahn-Str. 15, D-17489, Greifswald, Germany aut NATIONALLICENCE 700 1- Becher Dörte Institut für Mikrobiologie, Ernst-Moritz-Arndt Universität Greifswald, Friedrich-Ludwig-Jahn-Str. 15, D-17489, Greifswald, Germany aut NATIONALLICENCE 700 1- van Dijl Jan Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, P.O. Box 30001, 9700 RB, Groningen, The Netherlands aut NATIONALLICENCE 700 1- Buist Girbe Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, P.O. Box 30001, 9700 RB, Groningen, The Netherlands aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/21(2015-11-01), 9037-9048 0175-7598 99:21<9037 2015 99 253