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Expression Of Recombinan Human Lipoprotein Lipase Mutant Q402E In Pichia Pastoris

Posted on:2009-10-12Degree:MasterType:Thesis
Country:ChinaCandidate:X R XingFull Text:PDF
GTID:2120360278450431Subject:Internal Medicine
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Lipoprotein lipase (LPL) is a key enzyme which is involved in lipid metabolism. Tree shrew (TS) is an animal which is considered to be insusceptible to atherosclerosis(AS). Activity analysis showed the LPL activity in the medium transfected with the wild-type TS LPL is much higher than the wild-type human LPL. After constructed human LPL mutants based on the TS LPL sequence,we further compared the activity among Q402E mutants and wild-type human LPL.The results showed that activity of Q402E is much higher . In order to systematically investigate the mechanism, we select pichia pastoris X33 and made efforts to find a way to express LPLQ402E in different conditions in order to find a a effective way to express the recombination protein.Objective To construct yeast expression vector which can express human Lipoprotein lipase mutant Q402E and delivered into a Pichia pastoris strain X33. And then discusses the environmental factors such as the induction temperature,the concentration of methanol,time point ,and induction pH,which play an important role in afecting the yield and biological activity of the recombinant protein to produce large scale of active Lipoprotein lipase mutant Q402E .Methods LPL-Q402E cDNA had been inserted into pCDNA3.1 vector and was stored at -80℃in our Lab. So delivered into into chemically competent DH-5αE.Coli,the respective destination clones were selected by plating on ampicilin to obtain enough pCDNA3.1- LPL-Q402E recombinant plasmidwere. Then obtained Lipoprotein lipase mutant Q402E from pCDNA-Q402E by PCR and add restricted enzyme recognition site XhoI,XbaI on each side of LPL-Q402E.Restricted enzyme digest the pPICZαA and the PCR product to make adhesive end and then took T4DNAligase to cloned LPL-Q402E cDNA into pPICZaA .Delivered into into chemically competent TOP10F`Coli,the respective destination clones were selected by plating on Zeocin. Detect the recombinant plasmidwere by wall- primer,lateral-primer,restriction enzyme and Sequence analysis to confirm LPL-Q402E gene was inserted into yeast expression vector pPICZaC correctly.Linearize the plasmid prior to transformation and selection in Pichia. Transform the recombinant vectors into Pichia pastoris X-33 by Electroporation and screened each best strain in recombinant expression through Mut phenotype determination by MDH and MMH plates and YPDS plates containing 100μg/ml Zeocin? to isolate Zeocin?-resistant clones. Besides contain the presence of insert using PCR .After that started with expression induce by five percent of methanol.Collect 1 ml of the expression culture analyze the expression of our Protein by SDS-PAGE . Change the conditions such as temperature,concentration of methanol,time point to optimize expression.Results The expression plasmid pPICZαA- LPL-Q402E was constructed and the X33 cell strain which expressed LPL-Q402E in high level Was obtained.After 12 hours,we can see our protein by SDS-PAGE,and then increasing gradually until 72 hours.The hybridprotein increased rapidly after 48 hours . As the temperature fall down ,the hybridprotein was less than high temperature condition .Five percent of methanol was better than 1.0 % and 0.25%.Conclusion1.The LPL-Q402E yeast expression vector pPICZaA- LPL-Q402E is constructed successfully in the study.2. A method of expression of LPL-Q402E mutant in Pichia pastofis has been established.3. The recombination protein could be obtained at the maximum dose at 72 hours point.Lower temperature would be better for the expression of the recombination protein. The recombination protein could be express under 5% concentration of methanol which work as inductor.
Keywords/Search Tags:Recombinant Lipoprotein lipase Q402E mutant, pichia pastoris, expression
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