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Characterization Of Serpin-type Trypsin Inhibitor SW-AT-1 In Silkworm,Bombyx Mori

Posted on:2016-03-03Degree:MasterType:Thesis
Country:ChinaCandidate:Y HanFull Text:PDF
GTID:2310330512972806Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
Protease inhibitors regulate the function of proteases by inhibiting protease activity.Proteases play important roles in maintaining normal growth and disease resistance.Protease inhibitors distribute widely in plants,animals,bacteria,fungi and viruses.Serine protease inhibitor?SPI?is the most widely class of protease inhibitors.It is the major regulator of protease hydrolysis cascade.SPI regulating animals' coagulation,inflammation,tissue reparation and apoptosis life processes.The serine protease inhibitors also can defend against insects and pathogens,regulating of adversity excessive destruction of photolytic enzymes and increasing the antibacterial properties as well as insect resistance.According to the mechanism of serine protease inhibitor action,it can be classified as three categories:typical serine protease inhibitors,atypical serine protease inhibitors and serpin class.Typical serine protease takes the traditional lock-and-key model binding substrates,while the serpin inhibitors is covalently bound to a substrate and the active center of the annular zone by reactive centre loop?RCL?,forming a complex manner to produce irreversible inhibition effect.In our previous work,MALDI-TOF-MASS mass spectrometry methods have identified the trypsin inhibitor,silkworm antitrypsin isoform 1?SW-AT-1?,UniProt accession number was C7ASM9.C7ASM9 was named as silkworm antitrypsin isoform 1?GenBank:FJ613793?in the NCBI.Bioinformatics analysis of the protease inhibitor shows that it has 49 negative charge residues and 42 positive charged residues.It exists in silkworm without cross membrane area.Known from structural analysis of the SMART software,SW-AT-1 has a serpin domain?28-390 amino acid residues?and a signal peptide?1-16 amino acid residues?.Through the SW-AT-1 protein-structure prediction and bioinformatics analysis,RCL is a convex exposed region domain of the region.It is the critical area that controls the functions of SW-AT-1.RCL comprise of 10 amino acids:G327,A328,E329 and A330 located at N-end;and F352,N353,A354,N355,K356 and P357 located at C-segment.Construct mutagenesis is designed by site-directed PCR primers.The genes connect the prokaryotic expression vector pET-28a then getting pET-28a-SW-AT-1 and prokaryotic expression of mutant G327A,A328G,E329G,A330G,F352A,N353D,A354G,N355S,K356G and P357G The expression vector was transformed into an expression strain DE3 and large amounts of purified protein were gained by the Ni-NTA affinity chromatography.Through the enzyme kinetic analysis,calculated stoichiometry inhibitors of SW-AT-1 and mutants reacted with trypsin value 1.2,2.54,1.35,4.41,1.39,1.38,1.44,1.35,1.33,1.42,1.3 and SI of SW-AT-1,mutants reacted with chymotrypsin value 1.3,2.44,4.25,1.3,1.45,1.48,1.4,1.5,1.38,1.35.Ka of SW-AT-1,mutants reacted with trypsin value 1.62×105M-1S-1,0.89×105M-1S-1,1.66×105M-1S-1,1.88×105M-1S-1,1.53×105M-1S-1,1.68×105M-1S-1,1.43×105M-1S-1,1.45×105M-1S-1,1.54×105M-1S-1,1.66×105M-1S-1.Ka of SW-AT-1 and mutants reacted with chymotrypsin value 1.77×104M-1S-1,3.12×104M-1S-1,1.27×104M-1S-1,4.13×104M-1S-1,2.77×104M-1S-1,3.02×104M-1S-1,2.36×104M-1S-1,2.97×104M-1S-1,2.54×104M-1S-1,3.41×104M-1S-1.The stoichiometry inhibitors calculated the proportion of protease inhibitor and a substrate for the enzyme reaction.E329G and G327A mutants' SI values reached about three times of the wild-type,indicating that the 327,329 amino acids mutation in the RCL leading to inhibition of protease inhibitor reduced functionality.According to enzyme kinetics curves can be calculated protease inhibitor ka.The ka value of mutant G327A,E329G has large difference of WT.G327 and E329 amino acid residues are the key sites of RCL inhibitory activity.Construct the recombinant expression vector pCAMBIA-1304-SW-AT-1 and pCAMBIA-1304-ubi-SW-AT-1.Recombinant vector is transferred into rice suspension cells by agro bacterium infection method,and detection of total protein in rice suspension cells inhibit trypsin and chymotrypsin inhibitor activity.It indicates that the insertion of exogenous SW-AT-1 gene has an impact on the activity of proteins in plants.However,due to low amounts of exogenous protein expression,the activities of the recombinant proteins carrying different promoters have few differences.Expression of SW-AT-1 protease inhibitors in rice suspension cells can inhibit the intracellular protease activities and reduce the interference to other exogenous protease gene expressions.It provides a stable internal environment for the expressions of foreign genes in rice.
Keywords/Search Tags:serpin, trypsin inhibitor, chymotrypsin inhibitor, prokaryotic expression, site-directed mutagenesis, rice suspension cells
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