Secretory Expression,Purification And Activity Assay Of Recombinant Human FUT8 In Pichia Pastoris

Background:Core fucosyltransferase（FUT8）is the only glycosyltransferase that catalyzes the production ofα-1,6-linked fucose in mammals.The core fucosylation catalyzed by this enzyme plays a vital role in various life processes in the organisms,such as the maintenance of physiological homeostasis,B cell antigen recognition,intestinal microbiota,tumor microenvironment,etc.From a clinical point of view,FUT8 is closely related to atherosclerosis,abnormal schizophrenia,emphysema,tumor occurrence,development and outcome.Objective:The use of Pichia pastoris to express human FUT8 recombinant protein provides experimental materials for the study of the biological functions of FUT8.Methods:1.The editing of human FUT8 geneThe amino acid sequence of human FUT8 obtained from GenBank was edited by Snapgene software,removes the transmembrane region and part of the stem region of fut8 gene,and optimizes the remaining fragment according to yeast preference.Then insertα-factor at the amino terminal,the his-tag at the carboxyl,stop the codon,and add restriction endonuclease sites at both ends of the sequence.2.Construction of the recombinant FUT8 expression strain in Pichia pastorisThe recombinant FUT8 fragment was cloned into Pichia pastoris secretion vector pPICZαA by restriction endonuclease to form a recombinant plasmid pPICZαA-Fut8containing the Fut8 fragment,which was then electrotransformed into Pichia competent cells,and ice-cold Sorbitol helps yeast cells renaturation.Zeocin-containing plates were used to screen for successful transformation and recombinant FUT8 Pichia expression strains with zeocin resistance.3.Induced expression,purification and optimal enzymatic reaction conditions of recombinant FUT8The positive clone of recombinant FUT8 was inoculated into BMMY medium and induced by methanol.SDS-PAGE and western blotting were used to detected in the induced supernatant.The optimal enzymatic reaction conditions of recombinant FUT8were studied under different temperature and pH conditions.4.Activity detection and characterization of recombinant FUT8The activity of recombinant enzyme was detected by LCA,HPLC,and the heat tolerance of recombinant enzyme was detected by DSC（Differential Scanning Calorimetry）.5.Mass spectrometry methodGDP-L-Fucose,MES-NaOH（pH=7.0）and recombinant FUT8 were incubated with the serum of Fut8^-/-mice.Then the serum of Fut8^-/-mice,the serum of Fut8^+/+mice and the mixture mentioned above were released N-glcan by PNGase F and analyzed by mass spectrometry.Results:1.Construction of the recombinant FUT8 expression strain in Pichia pastorisThe results of restriction endonuclease digestion,agarose electrophoresis,and sequencing analysis showed that the FUT8 recombinant expression plasmid pPICZαA-Fut8 was successfully constructed,and the recombinant plasmid was Zeocin resistant.After electrotransformation of yeast cells,small white colonies grew on the plate containing Zeocin,which indicated that the recombinant plasmid pPICZαA-Fut8 was successfully introduced into Pichia pastoris X-33 strain.2.Recombinant FUT8 has enzyme activityThe activity of the recombinant enzyme was analyzed by LCA and HPLC,and the results showed that the recombinant FUT8 has good enzyme activity and catalyzesα-1,6fucosylation modification.3.The optimal enzymatic reaction conditions and characterization of recombinant enzymeThe enzyme activity of the recombinase was tested under different temperature and pH conditions.The results showed that the optimum temperature for the recombination FUT8 enzyme activity reaction was 37°C,and the optimum pH was 7.0.Recombinant FUT8 is heat-resistant and alkali-resistant.4.Mass Spectrometry analysisThe N-glycan in the samples released by PNGase F was analyzed by mass spectrometry.The results showed that theα-1,6 linked fucosylated N-glycan was detected in the serum of Fut8^+/+mice and the serum of Fut8^-/-mice+recombinant FUT8.Theα-1,6-linked fucosylated N-glycan was not detected in the FUT8^-/-mouse serum group.Conclusion:This experiment successfully established a method to secretedly express human FUT8 in Pichia pastoris.The human FUT8 was produced by this method is stable to heat and alkali and has high enzyme activity.