Versatile vector suite for the extracytoplasmic production and purification of heterologous His-tagged proteins in Lactococcus lactis
2015
Neef, Jolanda | Milder, Fin J. | Koedijk, Danny G. A. M. | Klaassens, Marindy | Heezius, Erik C. | van Strijp, Jos A. G. | Otto, Andreas | Becher, Dörte | van Dijl, Jan Maarten | Buist, Girbe
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.
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