Cloning, Expression Of The Key Enzyme Kfid From E. Colik5and Sulfating Modification Of Heparosan

Heparin and heparin sulfate have been widely used as anticoagulant drugs in clinical application, and may play an important role in anti-cancer and anti-virus. Pharmaceutical heparin is isolated from porcine, however, heparin, contaminated by oversulfated chondroitin sulfate, has a potential risk and unstable resources. Traditional chemical synthesis of heparin is costly and tedious, although this approach has successfully manufactured heparin. Therefore, synthesis of heparin and its derivatives from microorganism became a hot topic. This study includes cloning and expression of the key enzyme, and sulfating modification of heparosan from E. coli.First, a pair of specific primers was designed to amplify kfiD, The DNA fragment had100%identity with the kfiD from Escherichia coli Nissle1917and Escherichia coli ABU83972by BLAST alignment in NCBI. The gene fragment of KfiD encodes392amino acids with a molecular weight of44KDa.The gene fragment of kfiD was successfully cloned into expression vector pET28b. The resultant plasmid pET28b-kfiD was transformed into E. coli BL21(DE3). The effect of IPTG concentration and inducing time on the expression of KfiD was evaluated. Results demonstrated that the optimal IPTG concentration was0.4mmol/L and the optimal inducing time was4h.In addition, heparosan in the Escherichia coli K5culture was separated and purified by DEAE Sepharose FF with an average yield of12.7%.Sulfating modification of purified heparosan was conducted by concentrated sulfuric acid with different reaction time. After30min reaction, the sulfate content of heparosan sulfated derivatives was3.34%, and the degree of sulfur substitution was0.189. However, the sulfate content of the commercial heparin was18.25%, and the degree of sulfur substitution was2.2. Thus, heparosan need further sulfating modification via different methods to meet with the demand of final product.The structure of purified heparosan, heparosan sulfated derivatives, and commercial heparin were analyzed by1H-NMR.1H-NMR data showed that there existed indeed substitutions of sulfate in heparosan, in which sulfates located on GlcNAc (H1). Meanwhile, there might exist substitutions of sulfate at GlcA (HI), GlcNAc (H2, H3, H4, H5, H6), and GlcA (H2, H3, H4, H5, H6). However GlcNAc (CH3) would not be substituted. Moreover,1H-NMR of heparosan sulfated derivatives suggested there were not damage on the structure of disaccharide repeating units of heparosan.