Cloning And Expression Of Cbh2 In Humicola Insolens

Objective: Cellulose is the largest and renewable natual substance. It is stable chemically.It is very significant to use cellulose for the world energy crisis, food shortage, pollution problems.Cellulase is a kind of enzyme play a catalytic cellulose decomposition. The decomposition is high efficiency, convenience and low by-production. Investigation found that a lot of organisms like bacterias, actinomycetes fungi and insects etc can produce cellulase. People pay attention to the fungi because it can produce a variety of cellulases with high activity. Cellulase can be divided into three categories: endo-glucosidase, cellobiohydrolase,β-glucosidase.Domestic production of cellulase is very low. The most of cellulase we need relyed on import. People have tried a lot of methods to improve the activity and the production of cellulase. Traditional strain selection and combination kinds of cellulase can not increase catalysis of enzyme too much. For the high cellulase activity and stability, the use of gene engineering technology is significant. For example,altering the protein folding in active center by point mutation can increase the activity of enzymes. People also improve protein production by constructing engineering strains. And E.Coli, Saccharonyces cerevisiae and Pichia pastoris etc are used a lot as host cells. P pastoris has some special benefits in high production, easy cultivation, modifying protein like eucaryon. So it is in wide application.The thermophilic fungus Humicola insolen H31 was isolated from soil by the institution of Microbiology Chinese Academic of science in 1970. Humicola isolens H31-3 mutant strain is a good producer of thermostable neutral cellulase that can work in neutral condition. And Jiaji Shi mutanted Humicola insolens H31-3 to increase the enzyme production. The three major of extracellular neutral cellulase have been purified. The result of protein spectrum analysis is that H.insolens cbh2 is very similar with H.grisea O93780 in amino acids. But cellulase from fungy is homology. The nucleotide in active area and combination cellulose area is very similar. The same protein can have different gene if they come from different strain. If we want to develop cbh2, we should confine cbh2 gene first. The purpose of this experiment is to confine H. insolen H31-3 cbh2 gene. And express it in Pichia pastoris for a large number of cecretion.Method:1.Determin H insolen 31-3 cbh2 gene sequence1.1Cloning a part of cbh2 geneFirst, extract H.insolen's genome.Primer P1and P2 designing was based on the same nucleotide sequence with H.insolen cbh2 and H.grisea O93780. These primers can amplified 500bp nucleotide. The PCR production was purificated and sequenced.Comparative sequencing result with O93780 nucleotide by BLAST. The result is that this PCR production is the same as a part of O93780 nucleotide.1.2 Cloning full sequence of cbh2 and compare the production with O93780 P3 and P4 designing were based on the O93780 nucleotide which can cloning 1500bp gene fragment. After purification of the production ,it was cloned into pEASY-T vector. pEASY-T-CBH2 was transformed into Trans1-T1 Phage Resistant chemically Competent cell. Screening positive transformants by LB plate with ampiciline. After cultivation the transformants in small scale, plasmid was extracted.Sequence the positive plasmid which were verified by cloning with primer P3 and P4. Compare the sequencing result with O93780 nucleotide.The result is that the sequencing is the same as O93780.2.Combine two secretion vector; one is pGAPZaA-cbh2 cDNA which has vector's signal peptide, the other is pGAPZA-cbh2 cDNA2 which has cbh2's signal peptide.2.1 Extract H.insolens RNA.2.2 Cbh2 cDNA which isn't have it's signal peptide and cbh2 cDNA2 which has it's signal peptide were cloned.Primer 5 was designed to remove the O93780 native signal peptide sequence in the gene and generate a EcoR1 site. Primer6 was generated a Not1 site. Cbh2 cDNA was cloned by RT-PCR with P5and P6,and cbh2 cDNA2 was cloned with P3 and P4.2.3 Translate cbh2 cDNA and cbh2 cDNA2 into Trans1-T1 Phage Resistant chemically Competent cell to cloningAfter purification cbh2 cDNA and cbh2 cDNA2, they were cloned into pEASY-T vector. The pEAST-T-cbh2 cDNA and pEAST-T-cbh2 cDNA were transformed into Trans1-T1 Phage Resistant chemically. Screening and cultivation as the aforemention. PCR was used to determine positive transformants. Sequence the positive transformants. Compare the sequencing result with O93780 nucleotide.2.4 Combine two secretion plasmid and translate them into into Trans1-T1 Phage Resistant chemically Competent cell to cloning After purification cbh2 cDNA and cbh2 cDNA2, they were cloned into pEASY-T vector. The pEAST-T-cbh2 cDNA and pEAST-T-cbh2 cDNA were transformed into Trans1-T1 Phage Resistant chemically. Screening and cultivation as the aforemention.PCR was used to determine positive transformants. Sequence the positive transformants. Compare the sequencing result with O93780 nucleotide.3.Transform pGAPZaA-cbh2 cDNA and pGAPZA-cbh2 cDNA2 into Pichia pastoris to expression3.1 Preparation the P.pastoris competent cells3.2 Lined the recombimant pGAPZaA-cbh2 cDNA and pPGAPZA-cbh2 cDNA2 by BspH 13.3 Transformate the lined plasmid into P.pastoris competent cells. And screen the positive transformants by YPD which contain Zeocin antibiotic. Extract P.pastoris genome DNA.Identificate positive transformants by PCR. Sequenc PCR production with a-factor and 3-AOX to determine whether the intergration is right. 3.4 Confine cbh2 secretionSupernatant was collected and analyzed by SDS-PAGE. Compared with space bacteria, a gel slice which contained the expected protein was excised and analyzed by MS.3.5 Determine the optimum PH and temperature of cbh2.Detect the temperature stability of it.4. methanol induced cbh2 secretionIntergrated P.pastoris were cultured in BMGY until the strain OD=3, then collected it into BMMY and 1% methanol induced it expression.Determine cbh2 activity.Result:1. H.insolen H31-3 cbh2 has the same nucleotide as H.grisea O93780. These two protein have the same gene sequence.2. P.pastoris secretion the MW of 55KD cbh2.3. Only vector signal peptide can make cbh2 secretion.I am failed to secretion cbh2 in BMMY induced by methol.4. Optimum PH of cbh2 secreted by P.pastoris was 8 and Optimal temperature of it was 70℃. The activity of cbh2 is remain 65% after maintain at 50℃for 12 h.Conclusion:1.H31-3 cbh2 has the same gene with O937802.Humicola. insolens cbh2 with PH 8, optimum tempreture 70℃and MW 55KD cbh2 can be expressed and secreted by P.pastoris.