Expression Of Heparin Modification Enzymes In Insect Cells And Their Application In Oligosaccharide Synthesis

Heparan sulfate/heparin,a natural polysaccharide,is a linear glycosaminoglycan linked by uronic acid and glucosamine with 1-4 glycosidic bonds,and has biological activities such as antithrombotic,anticoagulant,antiviral,antitumor Since its discovery,it is still a research hotspot at home and abroad.The traditional heparin is prepared by extracting animal tissues as raw materials,and can be degraded into products of specific size or function.Considering that such animal-derived heparin has safety risks such as viral infection and the introduction of impurities,the development synthesis technology of non--animal-sourced heparin will be a milestone significance for its basic biological research and clinical application.In addition to simple chemical synthesis,there are two main ways to biologically synthesize non-animal-sourced heparin,one of which is started from heparin precursors obtained by fermentation of E.coli K5 as raw materials,modified by chemical methods and heparin modification enzymes to obtain heparin polysaccharide products.Another strategy is to synthesize the heparin backbone oligosaccharides de novo using UDP-GlcNTFA and UDP-GlcA as raw materials to,followed by enzymatically catalytic epimerization and sulfation using PAPS as the sulfate group donor to yield final heparin oligosaccharides with definite structure.The above biosynthetic methods have made progress,but limited in large-scale preparation of heparin mainly due to low yield,low activity and time-consuming preparation of heparin modification enzymes expressed in E.coli,which lead to incomplete catalytic reactions,byproduct formation and difficult to purify.In response to the problems,this paper focused on the expression of heparin-modified enzymes in insect cells and its application in oligosaccharide synthesis.The research contents and results are as follows.The C5-epi,HS2ST,HS6ST-1 and HPA genes were inserted respectively into plasmid pFastBacl by in-fusion cloning.The recombinant vector pFastBacl-C5-epi,pFastBac 1-HS2ST,pFastBacl-HS6ST-1,pFastBacl-HPA and positive control pFastBacl-GFP were transformed into E.coli DH10?Bac competent cells containing baculovirus shuttle vector and helper vector to obtain the recombinant Bacmid-C5-epi,Bacmid-HS2ST,Bacmid-HS6ST-1,Bacmid-HPA and Bacmid-GFP by the site-specific transposition that were confirmed by antibiotic resistance and blue-white screening.Then the recombinant bacmids were transfected into Sf9 insect cells via cellfectin to generate the recombinant baculoviruses expressing C5-epi,HS2ST?HS6ST-1 and HPA of interest.The culture supernatant of the infected Sf9 cells was analyzed by Western Blot for protein expression levels and determined for the enzymatic activity using synthetic heparin oligosaccharide substrate.The virus titer was determined by Reed-Muench.The time of infection(TOI)and multiplicity of infection(MOI)was optimized for the high expression of active C5-epi,HS2ST?HS6ST-1 and HPA.TCID50 of P3 virus was 10'6.75/0.01ml,and the pfu=108.6/ml.Then under different TOI conditions,the P3 recombinant viruses infected Sf9 cells,and the cell supernatants were collected for Western Blot identification and activity measurement.It was found that there was the highest protein expression level with low protein impurity in the supernatant of cells cultured for 72h,and the highest activity,so determining the optimal TOI of 72h.Then,Sf9 cells were infected with P3 recombinant viruses of different MOI for 72h,and the collected supernatant was subjected to Western Blot identification and activity measurement.It was found that when MOI was 1,the supernatant had the highest protein expression level and the best enzyme activity.In summary,the optimal condition for expression of the protein was 72 h after infection with MOI of 1.Using the introduced 6 × His tag at the N-terminus of the recombinant modification enzyme proteins was purified from the cell supernatant by Ni-NTA affinity chromatography to obtain C5-epi and HPA of 93%purity,HS6ST-lof 95%and HS2ST of 97%.Compared with heparin-modified enzymes expressed in E.coli respectively,the specific activity of the purified C5-epi enzyme expressed in Sf9 reached 118.57U/?g 27.32 times of that in E.coli.The specific activity of the purified HS2ST enzyme reached 29.38U/?g,1.31 times of that in E.coli.And the reaction catalyzed by HS2ST produced in Sf9 cells showed no by-products when heparin oligosaccharides are modified,while HS2ST expressed in E.coli led to 4 modified by-products.The specific activity of the purified HS6ST-1 enzyme reached 42.31U/?g,6.81 times of that in E.coli.Therefore,The recombinant human C5-epi with high activity was successfully expressed in Sf9,which provides an important enzymes for efficient synthesis and the large-scale production of the heparin and heparan sulfate.The crude UDP-Glc dehydrogenase was prepared by stepwise salting-out and heat denaturation and used to synthesize UDP-GlcA with a purity of 99.37%.The glycosyl donor UDP-GlcNTFA with a purity of 94.56%was synthesized using NahK,GlmU and PPA expressed in E.coli.Under the catalysis of glycosyltransferases KfiA and PmHS2,GlcNTFA or GIc A are alternately added to the non-reducing end of the starting material GIcA-PNP monosaccharide and its products to give the MS-certified heparin pentasaccharide backbone(GIcA-GIcNTFA-GIcA-GIcNTFA-GIcA-PNP)with a purity of 98.24%.After TFA group of the backbone was removed by chemical method,and it was further N-sulfated under the catalytic action of NST enzyme to obtain MS-verified GIcNS-GIcA-GIcNS-GIcA-PNP(referred to as 5mer-NS)at purity of 98.33%.Using 5mer-NS as a substrate,under the combined action of unpurified supernatant of insect cells expressing C5-epi and HS2ST enzyme,isomerization and sulfation modification were achieved in one step,and the product GIcA-GIcNS-IdoA2S-GIcNS-GIcA-PNP(referred to as 5mer-NS-IdoA2S)was verified by MS and had a purity of 98.81%.And then using 5mer-NS-IdoA2S as a substrate,the unpurified supernatant of insect cells expressing HS6ST-1 enzyme was used for 6-O-sulfation modification to obtain GIcA-GicNS6S-IdoA2S-GicNS6S-GIcA-PNP(referred to as 5mer-NS6S-IdoA2S)with a purity of 99.27%.Therefore,heparin-modified enzymes secretory expressed by insect cells do not require purification,and can efficiently and cost-effectively catalyze the modification of heparin oligosaccharides,which overcomes the drawbacks of heparin-modified enzymes expressed in E.coli and lays the foundation for large-scale synthesis of diverse heparin oligosaccharides.