Cloning and expression of the Lactococcus lactis subsp. cremoris SK11 gene encoding an extracellular serine proteinase.
The Lactococcus lactis subsp. cremoris SK11 plasmid-located prtP gene, encoding a cell-envelope-located proteinase (PrtP) that degrades alpha s1-, beta- and kappa-casein, was identified in a lambda EMBL3 gene library in Escherichia coli using immunological methods. The complete prtP gene could not be cloned in E. coli and L. lactis on high-copy-number plasmid vectors. However, using a low-copy-number vector, the complete prtP gene could be cloned in strains MG1363 and SK1128, proteinase-deficient derivatives of L. lactis subsp. lactis 712 and L. lactis subsp. cremoris SK11, respectively. The proteinase deficiency of these hosts was complemented to wild-type (wt) levels by the cloned SK11 prtP gene. The caseinolytic specificity of the proteinase specified by the cloned prtP gene was identical to that encoded by the wt proteinase plasmid, pSK111. The expression of recombinant plasmids containing 3' and 5' deletions of prtP was analyzed with specific attention directed towards the location of the gene products. In this way the expression signals of prtP were localized and overproduction was obtained in L. lactis subsp. lactis. Furthermore, a region at the C terminus of PrtP was identified which is involved in cell-envelope attachment in lactococci. A deletion derivative of prtP was constructed which specifies a C-terminally truncated proteinase that is well expressed and fully secreted into the medium, and still shows the same capacity to degrade alpha s1-, beta- and kappa-casein.
de Vos
W M
WM
Department of Biophysical Chemistry, Netherlands Institute for Dairy Research (NIZO), Ede.
Vos
P
P
de Haard
H
H
Boerrigter
I
I
D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005798:Genes, Bacterial; D005838:Genotype; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology / Q000235:genetics*; D010641:Phenotype; D010957:Plasmids; D015183:Restriction Mapping; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000235:genetics*
D012697:Serine Endopeptidases