Cloning And Expression Of Kfia For Heparosan Biosynthesis In E. Coli K5and Expression And Purification Of Heparinase Ⅱ

Heparosan, the main part of E. coli K5capsular polysaccharide, can be used as precursor for heparin biosynthesis. This study aims to improve heparosan yield by kfiA and kfiC overexpress in recombinant strain. A useful tool enzyme for the genetic engineering of E. coli K5, Heparinase II, was also expressed and purified.A kfiA fragment was cloned from the genomic DNA of E. coli K5via PCR technology. Then the fragment was inserted cloning vector pMD19-kfiA. After PCR identification, sequencing and Blast analysis of the resultant plasmid pMD19-kfiA, the kfiA fragment on the vector was proved100%similar to the target gene. Then expression vector for KfiA expression was constructed as pET28b-kfiA and transformed into E. coli BL21(DE3). KfiA was expressed best when inducted with an IPTG concentration of0.8mmol/L after4h culture time. After purified by denatured His-tag affinity chromatography and analysis by UN-Scan-it gel 6.1, it was proved that the expressed protein was the target protein.Then kfiC fragment was inserted to the vector pET28b-kfiA, and constructed the co-expression vector pET28b-kfiA-C in E. coli BL21(DE3). After PCR identification, double digestion, sequencing and blast analysis, the fragments on the vector was proved100%similar to the target gene. KfiA was expressed inducted with an IPTG concentration of0.8mmol/L, but the expression of KfiC was not clearly detected.Heparinase II was successfully expressed in auto-induction media by strain E. coli BL21(DE3)/pET15b-hepB. After nature His-Tag affinity chromatography, heparinase II was purified with purity96%, enzyme activity200U/mL, specific activity139U/mg. The yield of one-step purification was41.90%. Heparinase II was successfully used to eliminate heparosan; improve transformation of E. coli K5. The resultant recombinant strains E. coli K5/pET28b-kfiA and E. coli K5/pET28b-kfiA-C were successfully constructed. However, no significant target protein band was observed on SDS-PAGE gel of the recombinant strains culture after induction by IPTG. TEM result suggested that heparinase Ⅱ could eliminate capsule of E. coli K5.