The S-layer gene of Lactobacillus helveticus CNRZ 892:: cloning, sequence and heterologous expression
The S-layer gene of Lactobacillus helveticus CNRZ 892:: cloning, sequence and heterologous expression
复制标题
DOI：
10.1099/00221287-144-3-719
复制
发表时间：
1998-03-01
期刊：
影响因子：
2.8
通讯作者：
Morelli, L
中科院分区：
文献类型：
--
作者：
Callegari, ML;Riboli, B;Morelli, L
瑞士乳杆菌CNRZ 892含有由以规则阵列组织的43 kDa蛋白单体组成的表面层（S层）。已在大肠杆菌中克隆了编码该蛋白的基因（slpH）并进行了测序。slpH由440个密码子组成，前面是核糖体结合位点（RES），后面是推定的rho非依赖性终止子。事实上，北方分析揭示slpH是单顺反子基因。该基因之前是一个可能的启动子，其-35和-10六核苷酸间隔17个核苷酸。通过引物延伸分析，将转录起始位点定位在-10序列下游7 nt处，而推导的氨基酸序列显示30个氨基酸的前导肽。通过PCR扩增slpH基因，并将携带从RES到终止密码子的完整基因的片段克隆到启动子P32下游的乳球菌基因表达载体中。乳酸乳球菌MG 1363携带所得质粒，产生并分泌与真正的L. SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）结果表明，该菌株的蛋白质组成与瑞士黑曲霉CNRZ 892 SlipH一致。免疫电子显微镜显示，SLPH结合到乳球菌细胞壁中的小块和积累在生长培养基中的小片。
Lactobacillus helveticus CNRZ 892 contains a surface layer (S-layer) composed of protein monomers of 43 kDa organized in regular arrays. The gene encoding this protein (slpH) has been cloned in Escherichia coli and sequenced. slpH consists of 440 codons and is preceded by a ribosome-binding site (RES) and followed by a putative rho-independent terminator. Indeed, Northern analysis revealed that slpH is a monocistronic gene. The gene is preceded by a possible promoter of which the -35 and -10 hexanucleotides are separated by 17 nt. By primer extension analysis the transcription start site was mapped al 7 nt downstream of the -10 sequence while the deduced amino acid sequence of SlpH shows a leader peptide of 30 aa. The slpH gene has been amplified by PCR and the fragment, carrying the complete gene from the RES to the stop codon, has been cloned in a lactococcal gene expression vector downstream of promoter P32. Lactococcus lactis MG1363 carrying the resulting plasmid produced and secreted an S-layer monomer with the same molecular mass as the authentic L. helveticus CNRZ 892 SlpH, as judged by SDS-PAGE. Immunoelectron microscopy revealed that SlpH was bound to the lactococcal cell walls in small clumps and accumulated in the growth medium as small sheets.