Expression Of The Serine Protease Genes HaSP3,HaSP6 And HaSP24 From Helicoverpa Armigera (H(?)bner) In Escherichia Coli

Serine protease is the largest hydrolytic enzyme family in the organism,including trypsin,elastinase,chymotrypsin,thrombin and so on.They share the similar specific catalytic properties for the same active center composed by three key amino acid residues-serine,histidine and aspartic acid(Ser,His and Asp).They mainly engage in food digestion and absorption in organism,and also participate in lots of physiological processes,including development,signal transduction and blood coagulation.Serine proteases play an important role in Bt protoxins activation and degradation.After ingestion,Bt protoxins are turned to the smaller activated Cry proteins by the proteolysis of serine proteases.The activated Cry toxins bind to the specific receptors on insect midgut epithelial cells,insert into the membrane to form a pore on it and result in loss of membrance function,finally,the death of insect.Therefore,serine protease also plays an important role in developing resistance to Bt insecticidal proteins.It is proved that the lack of trypsin or low protease activity of midgut to reduce activation of protoxins,or increased expression of chymotrypsin to enhance degradation of activated toxins are responsible for the resistance to Bt.There is little known that which serine protease participates in activation of protoxins or degradation of toxins.With the mature of heterologous protein expression,it could be better to understand the role of a specific protease by using the expression products to do the proteolysis.In this study,the expression of three protease genes HaSP3,HaSP6,HaSP24 from Helicoverpa armigera was investigated in eight developmental stages and five tissues;then these genes were expressed successfully by optimizing the prokaryotic expression conditions;and finially,the expression products of HaSP3 and HaSP6 could show the biological activity to the general substrates,and Cryl Ac protoxin could be proteollytically degraded by rHaSP3.This prokaryotic expression system which is set up in this study is helpful to express more serine protease genes to do further study to understand the interaction between proteases and Bt toxins.1.Alignment of deduced amino acid sequences of HaSP3,HaSP6 and HaSP24 from H.armigera with other serine proteasesOur previous study found relative expression levels of HaSP3,HaSP6,HaSP24 in resistant strain SCD423 were up-regulated 2 times,1.43 times,4.6 times,respectively,compared to the susceptible strain.This up-regulation may leads to development of resistance.Here these three protease genes and other serine protease genes were aligned and analyzed.As putative trypsin and chymotrypsin,HaSP6 and HaSP3 have typical conserved motifs,including three catalytic triad residues(Ser,His and Asp),six Cys which form three disulfide bonds to maintain tertiary structure.However,for the substitution of His,one of the conserved catalytic residues,by Phe,HaSP24 is a typical nonproteolytic serine protease homolog.2.Expression profiles of HaSP3,HaSP6,HaSP24 in different developmental stages and different tissuesExpression patterns of HaSP3,HaSP6,HaSP24 were analyzed by RT-PCR in eight different developmental stages and five different tissues.The transcript expressions of HaSP3,HaSP6,HaSP24 in eggs,pupa and adults were very low,and there was no difference in male and female.Expression levels of HaSP3,HaSP6 in 2nd instar larva were about 1800,1000-fold higher than that in eggs.In contrast,HaSP24 was highly expressed in 5th instar larva,with 4000-fold higher than that in eggs.These three genes were highly expressed in the gut,foregut and midgut other than the other tissues.The expression levels of HaSP3,HaSP6,HaSP24 in foregut were 9.44,1.97,3.65-fold higher than that in other part of gut.3.Optimizing expression conditions of HaSP3 in Escherichia coli.A number of criteria must be considered when optimizing the expression conditions of a recombinant protein,such as host,fusion tags,temperature and duration of induction.We found the E.coli strain BL21(DE3)is an ideal host for the soluble expression compared to the E.coli strain BL21(DE3)pLysS and TransB(DE3).Then,the vector pET41a was confirmed that could help to reduce inclusion bodies compared to pET30a and pET32a.Finally,the temperature at 16? and duration of 22 hours were proved to be effective for expression.4.Expression of HaSP3,HaSP6,HaSP24 in Escherichia coli and proteolysis of CrylAc protoxin by rHaSP3The rHaSP3,rHaSP6,rHaSP24 were expressed in E.coli and purified by GstTrap affinity columns and HiTrap ion exchange columns.There is a 60 KDa which is proved to be GroEL by LC-MS/MS.The expression products of HaSP3 and HaSP6 could show the biological activity to the general substrates BApNA or SAAPFpNA with the activity 182.1×10-3.140.7×10-3mU/min/mg.Just as the result of amino acid analysis,rHaSP24 has no the proteolytic activity to the general substrates.After proteolysis by expression product of HaSP3 for 60 min,the band of Cryl Ac protoxin changed obviously,but we did not find active toxin forms at 65 KDa.It is proved that Cryl Ac protoxin could be proteollytically degraded by rHaSP3.Perhaps more pure and higher concentration of expression product or multiple proteases could achieve to activate protoxin.