[Cloning and expression of promoter and signal peptide function fragments from Lactococcus lactis in Escherichia coli].
Promoter and signal peptide function fragments from Lactococcus lactis were cloned in E. coli using a promoter-signal sequence probe vector pGPB14. Forty-two clones were obtained, whose level of resistance to ampicillin ranged from 100 to 8000 micrograms/ml. Eight clones were selected for beta-lactamase distribution assay. The beta-lactamase activity was found mainly in the periplasm, which indicated successful secretion of the enzyme. Southern hybridization test demonstrated that the inserted fragments were indeed from L. lactis. Restriction enzyme analysis revealed that the size of inserted fragments ranged from 80bp to 400 bp, four of which were sequenced on the vector pGEM-3Zf. It was found that the inserted fragments of pSEQ8 and pSEQ12 turned out to be part of the inserted fragments of pSEQ4 and pSEQ17 respectively. Promoter and translation initiation codon were found among all four fragments signal sequenced. One typical S. D. sequence and one atypical signal sequence were found in pHSB4 and pHSB8, while the other two contained no typical S. D. sequence and signal peptide sequence. In addition, it was found that the upstream regions of the promoter contributed to the efficiency of transcription initiation.
Huan
L
L
Institute of Microbiology, Chinese Academy of Sciences, Beijing.
Sun
H
H
Chen
X
X
Zhuang
Z
Z
Xue
Y
Y
D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics*; D013294:Lactococcus lactis / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics*; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D021382:Protein Sorting Signals