Expression Of Endothiapepsin-Like From Beauveria Bassiana In Pichia Pastoris And Detection Of Its Activity

Endothiapepsin,Endothia aspartic proteinase,is a proteolytic enzyme which isolated from the plant pathogenic fungus Endothia parasitica that causing parasitic diseases of Castanea mollissima.However,the endothiapepsin-like protein gene also exist in the entomopathogenic fungus Beauveria bassiana.In previous study,we found that the endothiapepsin-like protein and the other aspartic proteinases were all up-regulated in high-pathogenicity strain of B.bassiana GXsk1011 in infected the epidermis of silkworm,Bombyx mori,at early stage.But whether the protein playing a role in pathogenicity as well as other functions or not remain unknown.In the present study,the gene encoding endothiapepsin-like protein was amplified from B.bassiana GXsk1011 via PCR and was sequenced.Meanwhile,the amino acid sequences of endothiapepsin-like protein was aligned with orthologs endothiapepsin?CAA37944?from E.parasitica,endothiapepsin-like?XP008601085?from B.bassiana Bb2860,aspergillopepsin A-like aspartic endopeptidase?XP022385274?from Aspergillus bombycis and aspergillopepsin F?XP753324?from Aspergillus fumigatus used the MegAlign program of DNAStar 8.0.The results showed that the amino acid sequences of the five proteins have two Asp-Thr-Gly conserved domains at the N-terminus and the C-terminus,respectively.However,there was a Ser or Thr was mutated to Gly in the C-terminal conserved domain of B.bassiana GXsk1011.Subsequently,a series of questions were emerged.First,it is not clear whether the amino acid mutation in the C-terminal conserved domain of endothiapepsin-like protein has an effect on the activity in the B.bassiana GXsk1011,and what roles the protein play in infecting the epidermis of silkworm is unknow.Second,is it because of the amino acid mutation that the protein was named as endothiapepsin-like protein?Therefore,we try to express the endothiapepsin-like protein of B.bassiana GXsk1011in vitro used the Pichia pastoris expression system.The results are showed as follows:1.Cloning and bioinformatics analysis of BbeapA gene of removal signal peptideThe gene BbeapA of removal signal peptide was cloned and further analyzed by bioinformatics.First,two pairs of primers were designed based on the multiple cloning sites of vectors pPIC9K and pPICZ?A.Second,the RNA of B.bassiana GXsk1011 was extracted and reverse transcribed into cDNA.The gene BbeapA of removal signal peptide was successfully amplifihed with the template cDNA,and two pairs of primers were also used respectively.Multiple sequence alignment results indicated that the Ser/Thr existed in the C-terminal conserved domain of endothiapepsin-like protein from B.bassiana GXsk1011 was mutated to Gly.The molecular of 40 kDa of the endothiapepsin-like protein was predicted after increase of 6×His tag and yeast signal peptide as well as removal of a 16 aa signal peptide.The protein has an N-glycosylation site at position 134.The three-dimensional structure was successfully predicted using endothiapepsin from E.parasitica as template,and the protein has two conserved Asp residues at position 89 and 276,respectively.2.Expression of recombinant Endothiapepsin-like protein in Pichia pastoris.The endothiapepsin-like protein from B.bassiana GXsk1011 was expressed in P.pastoris used vectors pPIC9K and pPICZ?A,respectively.First,the recombinants P.pastoris GS115/pPIC9K-BbeapA and GS115/pPICZ?A-BbeapA were constructed for heterologous expression.Second,protein production was induced by addition of 100%methanol to a final concentration of 1%every 24 h.P.pastoris GS115/pPIC9K and GS115-pPICZ?A were also served as control groups.Then the fermentation supernatant of two recombinants including control groups were collected and detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis?SDS-PAGE?.The results showed that the two transformants harboring the gene BbeaPA produced the same two clearly visible bands with molecular mass about 40 kDa and 45 kDa in the SDS-PAGE gel,comparing with the control transformants harboring empty pPIC9K and pPICZ?A.The recombinants and control groups appeared the same results as described above by Western Blotting.So we choose the recombinant P.pastoris GS115/pPIC9K-BbeapA as follow-up experiment strain.The recombinant strain GS115/pPIC9K-BbeapA was cultured by BMMY with 1%methanol,and the induced sample was salted by 80%?NH₄?₂SO₄ overnight,precipitation was dissolved with 5 mL PBS and purified by Ni-NTA affinity chromatography,which showed the same two visible bands as described above.Interestingly,in the early stage of fermentation the visible band with molecular mass about 45 kDa is more obvious while the band 40 kD is blurred.However,the opposite result occurred in the late stage of fermentation,the band 40 kDa is obvious while the band 45 kD is blurred.Therefore,we speculated that the band of 45kD is caused by glycosylation of the recombinant protein,while the breaking of glycosylation chain of the protein results in the band of 40 kD in SDS-PAGE gel.3.Detection of recombinant Endothiapepsin-like enzyme activityTo measure recombinant endothiapepsin-like protein activity,the yeast transformant GS115/pPIC9K-BbeapA were induced by 1%?v/v?methanol at 28?for 4days,and the supernatant of culture was collected and purified by Ni-NTA affinity chromatography.First,with casein as a substrate,the activity of recombinant endothiapepsin-like protein was 83.55 U/mg when the temperature was 40?and the pH value was 4.0.Second,we used the epidermis of silkworm as substrate to detect the ability of recombinant protein releasing the cuticle,which showed that the amount of proteins released from epidermis of silkworm by recombinant endothiapepsin-like protein was 1.4-fold higher than the control PBS?P<0.01?.In addition,the released proteins of silkworm cuticles were quantitatively tested by SDS-PAGE gel.We viewed the visible bands with molecular below 10 kDa were clustered.The epidermis of silkworm treated with the recombinant endothiapepsin-like protein was further analyzed by scanning electron microscopy.We found that the surface gully of the epidermis treated with recombinant endothiapepsin-like protein was deeper compared with the control,and the surface become cleaner and smoother.These results indicated that the endothiapepsin-like protein from B.bassiana GXsk1011 has activity and the protein can degrade the epidermis of silkworm.We further speculated that the endothiapepsin-like protein may be involved in the infection process of the insect by B.bassiana.