Functional Study Of Key Amino Acids In E2 Protein Of Recombinant Side Chain Dioxygate Dehydrogenase Complex

Side chain dioxygate dehydrogenase complex E2 protein(BCOADC-E2)can specifically react with anti mitochondrial antibody M2 subtype(AMA-M2).The amino acids around the active center of BCOADC-E2 protein were mutated into alanine by "alanine scanning",thus forming a series of mutant proteins.By comparing the specific reaction degree of each mutant protein and wild-type protein with AMA-M2,the function of amino acids replaced by alanine was explored,and the key amino acids of BCOADC-E2 protein were screened.In this study,we cloned the homologous target gene sequence BCKD which can express the antigen epitope of BCOADC-E2 protein in vitro,and recombined it with the engineering plasmid pGEX-4T1 to obtain the recombinant plasmid pGEX-BCKD.The recombinant plasmid pGEX-BCKD was used as a template,and the sequence was amplified by overlap extension Fifteen point mutation plasmids were obtained by site directed mutagenesis technique.Each plasmid was transferred into prokaryotic expression strain for protein expression.Fourteen mutant proteins and one wild-type protein were obtained by protein isolation and purification.The degree of specific reaction between 14 mutant proteins and AMA-M2 was determined by indirect ELISA The amino acids that have key influence on the specific reaction of BCOADC-E2 and AMA-M2 were identified.The main conclusions of this study are as follows:(1)the recombinant plasmid pGEX-BCKD was successfully constructed by gene sequencing;15 point mutation plasmids were successfully mutated into the expected genes by gene sequencing,and the rest of the point genes remained unchanged,indicating that all the 15 point mutation plasmids were successfully constructed;(2)the original plasmids containing all plasmids were successfully constructed protein expression,cell disruption,centrifugation,purification,and twelve sodium sulphate polyacrylamide gel electrophoresis showed that 14 mutant proteins and 1 wild type proteins were expressed in prokaryotic expression strains,and the purity and purity of the 15 proteins reached the requirement of determination;(3)The degree of specific reaction between mutant protein pGEX-BCKD-S1A and mutant protein pGEX-BCKD-C3A and AMA-M2 was higher than that between wild-type protein pGEX-BCKD and AMA-M2,which indicated that serine at position 1 and cysteine at position 3 of BCOADC-E2 were the key amino acids to enhance the specific reaction between BCOADC-E2 and AMA-M2.(4)The degree of specific reaction between mutant protein pGEX-BCKD-E4A,pGEX-BCKD-V5A,pGEX-BCKD-Q6A,pGEX-BCKD-S7A,pGEX-BCKD-D8A,pGEX-BCKD-S10A,pGEX-BCKD-V11A,pGEX-BCKD-T12A,pGEX-BCKD-113A,pGEX-BCKD-T14A,pGEX-BCKD-S15A,pGEX-BCKD-R16A and AMA-M2 was lower than that between wild-type protein pGEX-BCKD and AMA-M2,indicating that the amino acids of BCOADC-E2 were glutamic acid at position 4,valine at position 5,glutamine at position 6,serine at position 7,aspartic acid at position 8,serine at position 10,valine at position 11,threonine at position 12,isoleucine at position 13,threonine at position 14,serine at position 15 and serine at position 16 The mutant protein formed by the substitution of arginine with alanine can reduce the specific reaction with AMA-M2.(5)The average OD450 values of the mutant proteins pGEX-BCKD-V5 A and pGEX-BCKD-I13A were less than 1/10 of the control values,indicating that the substitution of valine at position 5 and threonine at position 13 of BCOADC-E2 protein by alanine would greatly reduce its specific reaction with AMA-M2,which indicated that valine at position 5 and isoleucine at position 13 are the key amino acids that affect the specific reaction between BCOADC-E2 and AMA-M2,and play an important role in the function of BCOADC-E2 protein.