Cloning, Expression Of Kfic From E. Coli K5and Depolymerization Of Heparosan

Heparin, a polysaccharide, is widely used as an anticoagulant drug.Heparosan，the capsular polysaccharide of Escherichia coli K5strain,serves as the critical precursor for the prepatation of bioengineered heparin.This study aimed at producing heparosan with a high yield through cloningof KfiC from E. coli K5and its inducing expression. Besides, comparedwith heparin which exhibits some undesirable side effects when used as aclinical anticoagulant, low molecular weight heparins (LMWH) tend to bethe heparin substitutes which have reduced side effects, more predictablepharmacological action, sustained antithrombotic activity, and betterbioavailability. In this study, the methods and conditions of heparosande-polymerizing to prepare the low molecular weight heparosan wereevaluated. It is useful for the potential application of low molecular weightheparosan for synthesis of LMWH.Firstly, kfic was successfully cloned using PCR. It was found the kfic 100%homology to that of the Escherichia coli strain Nissle1917, aftersequence comparison using BLAST on NCBI. The kfic was cloned into theexpression vector, pET28b. The resultant expression plasmid pET28b-kficwas transformed into E.coli BL21(DE3).The effect of polyacrylamide gel concentration and sample volume onheparosan PAGE result was evaluated. Results demonstrated that theoptimal polyacrylamide gel concentration was10%and the best samplevolumes were1.5mg rude heparosan,1mg pure heparosan, and0.06mgcommercial heparin.The effect of various de-polymerization methods, biological, chemical,and physical depolymerization of heparosan, was evaluated. The resultsshowed that heparinase II (Hep II) method was the best to effectivelyde-polymerize heparosan, of which the best buffer for the enzymatichydrolysis was buffer B (100mM sodium acetate,0.01%BSA and10mMcalcium acetate, pH7.0), the best dose of Hep II was19U/mL and the bestreaction time was1.5h. Additionally, H2O2-acetic acid method also showedobvious de-polymerization effect on heparosan, of which the optimumconcentration of H2O2and acetic acid was4.5%and6%, respectively, thebest temperature was55℃and the optimum reaction time was5h.Heparosan can also be de-polymerized by ultrasonic method, by which themolecular weight of the product was gradually reduced when the treatmenttime increased. The efficiency of the ultrasonic de-polymerization was enhanced by adding NaNO2.