The Expression And Refolding Of Nattokinase

Nattokinase produced by the nattokinase gene in Bacillus subtilis,is obtained from the traditional Japanese soybean food natto,and found to be a strong and safe fibrinolytic enzyme.The controversy always exists in the expression of nattokinase whether the nattokinase,as the inclusion body,can refold correctly through the regular refolding methods,without the help of the leading peptide,both of which give little hint about the specific refolding methods of nattokinase.Through analysing the previous expression methods of nattokinase,we put forward the question whether the issue lies in the various redundant peptides,which might give rise to the distinct influences on the refolding.Given the issue above,the expression vector pET-42b-NK was constructed with NdeI and XhoI, transformed into BL21,and induced by IPTG to produce the nattokinase as the inclusion body.Basing on the characteristics of pET-42b,the C terminal of the expressed nattokinase was binded with His-Tag to facilitate the purifying and refolding,at the same time the redundant peptides of the N terminal was removed to minimize its possibly bad effect on the refolding.After the nattokinase from the inclusion body was denatured by urea,and purified through affinity chromatography,the refolding methods of dialyzing and diluting with the addition of various refolding additives,were applied to refold the nattokinase to study that without the help of the leading peptide and the influence of the redundant peptides of the N terminal,whether the regular refolding methods and refolding additives can refold the nattokinase correctly,to give an controversy of some kind about the lasting issue.The results were as follows:1.The expression pET-42b-NK vector was constructed successfully,with the C terminal of nattokinase binded with His-Tag,and the N terminal not with any redundant peptides.2.The highly effective expression strain of E.coli.BL21 and inductive method were obtained to produce nattokinase as the inclusion body,which accounts for 30%of the total proteins.3.A set of methods about isolating the inclusion body and purifying with affinity chromatography were established to obtain the highly pure protein wih only one step.4.The study of the refolding of nattokinase indicates that the regular refolding methods and refolding additives cannot refold the nattokinase correctly,without the help of the leading peptide and the influence of the redundant peptides of the N terminal,which gives support to the opposite argument. In order to obtain the nattokinase with high production and strong activity,other methods were needed,such as expressing the leading peptides to work as an additive,or mutating the nattokinase through error prone PCR to get the mutant,which can refold correctly without the help of the leading peptides.