Expression and purification of Ebola virus trimeric glycoprotein based on novel glycoengineered Pichia pastoris / 军事医学

ABSTRACT

Objective To express and purify mammalian glycosylation modified trimeric Ebola virus trimeric glycoprotein (EBOV-GP) using novel glycoengineered Pichia pastoris.Methods The EBOV-GP and EBOV-GPΔMLDΔTM genes were cloned into the pPICZ-αA vector,electrochemically converted to glycoengineered Pichia pastoris,and compared with EBOV-GP expressed in HEK-293T cells.Glycosylation was analyzed by PNGaseF and EndoH digestion,while the target protein was purified by affinity chromatography and ion exchange chromatography.N-terminal sequencing was used to determine whether the signal peptide was correctly cleaved during protein translation and gel column analysis was used to find out whether the trimeric structure was formed.Results The results of PNGaseF showed that EBOV-GP expressed by glycoengineered Pichia pastoris and HEK-293T cells had the same relative molecular mass and N-glycosylation degree.EndoH digestion showed that the N-glycosylation modification of EBOV-GPΔMLDΔTM was in a non-high mannose form.N-terminal sequencing showed that the signal peptide of the GP protein itself was correctly excised.Gel column analysis showed that the purified protein was in a trimeric form.Conclusion An EBOV-GP is obtained with complex glycosylation modification based on Glycoengineered Pichia pastoris.