Streptomyces Griseus S106 Endopeptidase Gene Cloning And Prokaryotic Expression

Streptomyces griseus S106 and Streptomyces globisporus S186 possessing high lytic activity against Streptococc mutans and Staphylococcus aureus was isolated from soils by the double layer plate containing intact cells of Streptococcus mutans. We have cloned the N,O-diacetylmuramidase gene from the S106 genome. Lysozyme can classified into muramidase, endopeptidase, N-acetylmuramoyl-L-alanine amidase etc. which not only have bacteriolytic, but also play critical roles in cell growth. So we want clone the endopeptidase or N-acetylmuramoyl-L-alanine amidase gene sequences, express them in E.coli and research their enzyme chracterictics.We have acquired many endopeptidase sequences and N-acetylmuramoyl-L-alanine amidase sequences of Streptomyces from NCBI protein database, and aligned these sequences by Clustw respectively, then selected the suitable conserved amino acids based the CDD database, designed the CODEHOP primers online, the partial endopeptidase sequences and partial amidase sequences were amplified by Touchdown PCR from Streptomyces griseus 106 and Streptomyces globisporus 186 genomic DNA .the amplification products were ligated to pUcm-T vector, and transformed into E.coli DH5a, the positive cloning was sleceted and identified .The nest primers were designed based the acquired partial endopepteidase sequences, and the 5' and 3' sequences adjoin to the partial endopeptidase sequences of Streptomyces griseus 106 were amplified by TAIL-PCR, An opening reading frame,designated pR4,of 711bp that encoded a polypeptide of 237 amino acid residues was identified, the blastp results indicated that the pR4 gene was one of the members of the peptidase 23/37 family, showed 69% identity with a peptidase from the Streptomyces coelicolor, we also analyed the basic property of the mature endopeptidase by the bio-softwares, the analyzed results indicated the the pR4 gene was composed of a 39 amino acid residues signal peptide and a 198 amino acid residues mature polypeptide which was designated as R4 gene, the pI and MW of the R4 gene were 9.57 and 19971.10Da, we also constructed the phylogenetic tree of 50 peptidase sequences from other strains.The R4 gene was amplified by a pairs of primers from the Streptomyces genomic DNA, A recombinant plasmid pET-26b(+)-R4 was contructed by inserting the PCR product into pET-26b(+) vector and transformed into E. coli BL21 (DE3) plySs, after being analyzed by colony PCR and sequencing, it was confirmed that heterogeneous gene was inserted the recombinant vector, the recombinant protein was highly expressed after IPTG induction, and analyzed by the SDS-PAGE. The enzyme activity research showed that endopeptidase R4 have low activity on rupturing the S. aureus and Streptococcus mutans, but this phenomenon may caused by the unsuitable substrate and reaction condition.We also amplified the upstream sequences of R2 gene, the upstream sequence acquired by TAIL-PCR included partial oxidoreductase gene which encode 240 amino acid residues, 77 amino acid residues of R2 signal peptide and 302bp interval sequence between two ORFs. The 302bp sequence wasn't found obvious promoter components, further reseaech was needed to identify whether it possessed promoter activity.