Bacillus Extracellular High Abundance Protein Separation Sequencing, Cloning And Expression Of Components

The objective of this research is to clone the expression element of a major secretory protein gene from Bacillus sp.JK1.The strain in this research was isolated from fungi that treated blue-green algae on Taihu Lake.Identificated by morphological characteristics,biolog gram-positive(GN) assay and 16S rRNA gene amplification.Because of the incertitude result of biolog,the strain was designated as Bacillus sp.JK1.In the same time,two strains isolated from rabbit excreta were identificated as Bacillus megaterium RF3 and Bacillus megaterium RF5.SDS-PAGE analyzing the secretory proteins of Bacillus sp.JK1 showed that there were two major bands.One was smaller than 14.4KDa;another was about 18KDa, marked as P1 and P2,respectively.Transformed P1 and P2 to PVDF membrane and took N- terminal sequencing.P1 sequencing unsuccessfully.Result of P2 was as follows: ASVEDFSNYE.After NCBI blast,no homologous protein was found.RSD-PCR was used to cloning upstream and downstream sequences.Eight specific fragments were cloned,marked as U1,U2,U3,U4,U5,U6,U7 and D1,respectively.But no one was aim.Fragment U3 was biotin synthesis protein.U5 was NrdG and QueF.Both of fragments U6 and U7 was CrcB.Fragment D1 showed 100%amino acid identity with osmotic shock protection protein.The whole omsY is 685bp,and calculated protein weight is 23KDa.Sequence analysis shew there was a signal peptide in the N- terminal.The sequence "SNYE" in signal peptide was the same as "SNYE" in result of P2 N- terminal sequencing.