Study On Heterologous Soluble Expression And Characterization Of Heparinase ? In Prokaryotic Engineering Bacteria

Heparinase,derived from Flavobacterium heparinum,is a kind of polysaccharide lyase which can specifically degrade heparin and heparan sulfate.At present,it has been found that heparinase plays an important biological role in preparing low molecular weight heparin,inhibiting tumor cell proliferation and removing residual heparin in blood.At present,heparinase I is the most studied.Heparinase ? has low soluble expression in recombinant Escherichia coli,which leads to its low yield,difficult purification,high cost and other problems,which limits its application in medicine.Therefore,it is of great significance to find suitable methods to improve the yield and purity of heparinase I.Chaperonin CpkA is a thermostable chaperonin protein found in thermophilic bacteria Thermococcus kodakarensis.It has auxiliary protein folding function and ATPase activity.In this paper,the recombinant plasmid pACYC-CpkA and HepI'-28 a plasmid with cut-off signal peptide and positively charged signal-tag were constructed and transformed into E.coli DE3(BL21)in the form of single transfer or co-transformation.The soluble HepI' protein was obtained by nickel column purification,desalination column desalination and cation exchange column chromatography.At the same time,the prokaryotic expression of chaperonin CpkA,in E.coli DE3-RIPL was heat treated at 85? for 1 h,precipitated with ammonium sulfate with 80% saturation,desalted by dialysis and anion exchange column chromatography to obtain high purity CpkA protein.The chaperonin CpkA protein prepared in this paper was used for urea gradient renaturation with HepI inclusion body protein in vitro.The results showed that after the signal peptide was removed,the soluble expression of heparinase I increased significantly,and the inclusion body protein was further reduced after co-expression with CpkA.Finally,the final purification yielded a protein with a size of 43 kDa,a purity of more than 90%,a maximum concentration of 0.404 mg/ml of HepI' protein,and an enzyme activity of 27.16 IU/mg;A CpkA protein of 59.17 kDa,with a purity of 93% and a concentration of 2.706 mg/ml was obtained by prokaryotic expression,and its ATPase activity was 539 IU/mg;The purified CpkA protein was used to assist the urea gradient renaturation of Hep I inclusion body protein in vitro,the recovery rate of inclusion body protein was about 50%,and the renatured protein had no obvious heparinase I activity.This study provides a new method for the preparation of heparinase I and lays a material foundation for the wide application of heparinase I in medicine.It also provides a new experimental basis for chaperonin CpkA to help heterologous protein folding in vivo.