Expression Of Biotin Ligase BirA In Escherichia Coli Rosetta Blue And Pichia Pastoris KM71

Biotin ligase(BirA)is a protease widely exists in prokaryotes and eukaryotes,It's also called biotin holoenzyme synthetases,BirA enzyme,Biotin acetyl coenzyme A synthetase.It is an enzyme that is necessary for an organic life to obtain an essential vitamin-biotin.The basic principle for organisms to obtain the needed biotin is to activate biotin to be biotin-5'AMP,then transfer biotin to the corresponding receptor under the exist of ATP.In addition,due to the extremely high affinity between the biotin and streptavidin,which is a million higher than the affinity between antibody and antigen,they have been used to construct Biotin-Avidin—System.By combing with the commonly used marker enzymes(HRP or AKP),antibody(one or two anti),separation and purification tags(GST,His,etc.),BAS has been widely used in the qualitative and quantitative detection and localization of micro antigen antibody,the labeling and sorting of monoclonal antibodies,the labeling,separation and purification of a variety of proteins.It has a series of advantages such as high sensitivity,strong specificity,good stability,wide universal,simple manipulation and observation etc.BAS has now become one of the most important methods to study the interaction between protein and protein.At present,the domestic used BirA biotin ligases in the study of biotin avidin interaction are from prokaryotes.Escherichia coli expression system is the most commonly used method,which got the induced expression of the target protein by adding IPTG to the culture.Although Escherichia coli expression system has a series of advantages such as the short incubation period,recombination stability,simple culture method ect.Compared to the Pichia pastoris expression system,it also has some defects:easy to from inclusion;too much background protein;low expression;difficult to purification;high protein lost.Because of the defects mentioned above,it's difficult for the the target protein to get large-scale industrial production.So far,the expression of BirA using Pichia pastoris as the expression system has not been reported.The present study obtain the bira gene sequence from the NCBI database first,and design the corresponding primer and revise primer,then got the bira gene pcr products using E.coli rosetta blue as the template.After that,the target gene was inserted into expression vector pet-23a.On the other hand,a fusion protein nusa sequence was added to the 5'terminal of the target gene sequene and insert into vector Ppic9k as a whole.Finally,the two constructed vectors mentioned aboved were fransfer into E.coli rosetta blue and Pichia pastoris KM71.We successfully got the expression of BirA in Escherichia coli and Pichia pastoris eapression system.The shake flask yield of BirA in E.coli rosetta blue is 78 mg/L,the shake flask yield of BirA in Pichia pastoris KM71 is 103mg/L,and the fermentation expression of BirA reach 583 mg/L.The produced BirA ligase in both expression system was showed to be highly active in catalyzing the in vitro biotinylation.