Cloning, Prokaryotic Expression and Functional Analysis of pep Gene from Bacillus thuringiensis Lysogenic bacteriophage

Abstract:

[Objective] Random outbreak of lysogenic bacteriophage from Bacillus thuringiensis was very harmful to the production of Bacillus thuringiensis insecticide. We clarified the background of the phage from Bacillus thuringiensis MZ1 at the molecular level to solve the problem of random outbreak. [Methods] After the strain MZ1 from a company in Meixian County of Guangdong Province was induced, we obtained phage particles. Then phage DNA was extracted and pep gene was cloned, expressed and analyzed. [Results] We obtained a lysogenic bacteriophage MZTP01with clear plaque and 1 mm diameter. Fragment D with 2362bp (Genebank No. AY639599) was obtained after the phage DNA hydrolyzed by HindⅢ/EcoRⅠ. Among fragment D, the pep gene with molecular weight of 47 kDa and length of 1101bp was cloned and expressed. Recombinant M15 (pQE30pep) was built and overexpressed in Escherichia coli with a 47kDa clear band. At the same place a clear band was observed by Western blot. Judging from the time course expression, we could conclude that PEP protein produced at 1 hour after induction and then increased gradually. PEP protein was mainly in the form of inclusion body in the recombinant and slowed the growth speed of host. Homologous comparison of PEP protein from phage MZTP01 with other PEPs from BLAST were that phage MZTP01 PEP protein had 100% homologe with that of Escherichia coli K12, and most of others took the similarity in the range between 37%~84%. [Conclusion] PEP protein had ability to hydrolyze casein with the enzyme activity of 0.3mg/ml trypsin. PEP protein may be a kind of trypsin.