The Expression, Purification And Directed Evolution In Vitro Of Anthrax Toxin

Anthrax caused by Bacillus anthracis is a zoonosis disease which led to great hazard to human heath and stockbreeding.Because of high mortality and strong vitality of the spore of Bacillus anthracis,it is used as bioweapons and bioterrorism that lead to turbulence in society.So it is very important to research anthrax.The anthrax toxin is a binary bacterium toxin composed of protective antigen（PA）,lethal factor（LF） and edema toxin（EF）.PA moiety is a core factor for anthrax-related research and the common way to obtain PA is to express the recombinant PA in E.coli.However,the recombinant PA was an inactive inclusion body form expressed in E.coli and multi-procedures are needed to purify the protein.In this study,we found that PA solubility enhanced dramatically when fused with Glutathione S-transferase（GST） and induced at 28℃and 16℃.The PA was purified with high homogeneity and a yield of 3 mg protein was obtained from 1 liter culture by the affinity chromatography approach.Moreover,we expressed and purified three PA mutants,I394C,A396C and N435C,which were not expressed in E.coli reported preciously.The cell cytotoxicity assay showed N435C was a novel inactive defective mutant which conferred dominant-negative inhibitory activity.That may be useful in designing new antitoxin for anthrax therapy.On the other hand,we constructed random mutation library of the 774 bp fragment of lethal factor from 1550 to 2324 bp（517-776 aa） to study the pathogeneisis of lethal factor and therapy and prophylaxis against anthrax more effective.We scanned the library by detecting the supernatant activity of LF mutations lysate.As a result,we got five mutants which had low biological activity.Furthermore,we purified two mutant proteins only carrying one mutate site and got the two mutant proteins K518E and L519C for the first time.The cytotoxicity assay showed that the two mutant proteins lost activity significantly,that means,Lys⁵¹⁸ and Leu⁵¹⁹ played an important role in the function of lethal factor.Further,K518E endowed with the character of competition inhibition of wide type LF which may be helpful to the study of lethal mechanism.Probably,mutant K518E change Lys into Glu carrying negative charge and affects the catalytic groove of LF which is negative charge.Therefore,Lys 518 maybe has an important role to stabilize the conformation of LF and combine the substrate.