Geller B L, Wade N, Gilberts T D, Hruby D E, Johanson R, Topisirovic L
Department of Microbiology, Oregon State University, Corvallis, Oregon 97331-3804, USA.
Appl Environ Microbiol. 2001 Dec;67(12):5370-6. doi: 10.1128/AEM.67.12.5370-5376.2001.
The C repeat region of the M6 protein (M6c) from Streptococcus pyogenes was expressed within the Pip bacteriophage receptor on the surface of Lactococcus lactis. M6c was also detected in the culture medium. The pip-emm6c allele was integrated into the chromosome and stably expressed without antibiotic selection. The level of cell-associated surface expression of PipM6c was 0.015% of total cellular protein. The amount of PipM6c on the cell surface was increased about 17-fold by expressing pip-emm6c from a high-copy-number plasmid. Replacing the native pip promoter with stronger promoters isolated previously from Lactobacillus acidophilus increased surface expression of PipM6c from the high-copy-number plasmid up to 27-fold. Concomitantly, the amount of PipM6c in the medium increased 113-fold. The amount of PipM6c did not vary greatly between exponential- and stationary-phase cultures. Western blots indicated that the full-length PipM6c protein and most of the numerous proteolytic products were found only on the cell surface, whereas only one proteolytic fragment was found in the culture medium.
化脓性链球菌M6蛋白(M6c)的C重复区域在乳酸乳球菌表面的Pip噬菌体受体中表达。在培养基中也检测到了M6c。pip-emm6c等位基因被整合到染色体中,并且在没有抗生素选择的情况下稳定表达。PipM6c与细胞相关的表面表达水平为总细胞蛋白的0.015%。通过从高拷贝数质粒表达pip-emm6c,细胞表面PipM6c的量增加了约17倍。用先前从嗜酸乳杆菌分离的更强启动子替换天然pip启动子,可使高拷贝数质粒的PipM6c表面表达增加至27倍。与此同时,培养基中PipM6c的量增加了113倍。在指数生长期和稳定期培养物之间,PipM6c的量变化不大。蛋白质免疫印迹表明,全长PipM6c蛋白和大多数大量的蛋白水解产物仅在细胞表面发现,而在培养基中仅发现一个蛋白水解片段。