资源描述
单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,2014/7/22,#,Literature report,Content,1. High-level expression of the native barley,-amylase/subtilisin inhibitor in Pichia pastoris,2. A Manual for the Preparation and Transformation,of Pichia pastoris Spheroplasts,High-level expression of the native barley,-amylase/subtilisin inhibitor in Pichia pastoris,Introduction / Progress,1.,Materials and methods,2.,Results,3.,Discussion,4.,Content,Back,1.Introduction/ Progress,(1),Previously,most plant proteins expressed in P.pastoris resulted,in low yields,(Cereghino and Cregg,2000,).,(2),BASI,(the Hordeum vulgare,-amylase/subtilisin inhibitor ),has,previously,been expressed in P. pastoris , but,yields,were very,low,(,Bnsager et al,.,2003,).,(3),Pichia pastoris,is one of the,eukaryotic expression hosts,that have been,developed,into,a highly successful expression,system,(Macauley-Patrick et al.,2005,).,1.Introduction/ Progress,(4) However,within the last couple of years, plant proteins have,successfully been expressed,in P. pastoris,at high levels,(Fierenset al., 2004; Yoneyama et al.,2007,; Pan et al.,2007,).,(5),In this study,we present,successful high-level expression of,the Hordeum vulgare,-amylase/subtilisin inhibitor,(BASI),in P.pastoris,. (From,Journal of Biotechnology,2008,),(6),In this paper,a high-level expression system for the,bi,functional,-amylase/subtilisin,inhibitor,from barley,is presented.,1.Introduction/ Progress,(7),Compared to,previous attempts to express BASI in P. pastoris,(,Bnsager et al., 2003,),various conditions,have been,optimized in this study, including,signal sequence,codon usage,transformation technique,and,growth conditions,.,Back,2. Materials and methods,2.1. Strains, culture media and growth conditions,(1),P. pastoris,(,Invitrogen, USA) GS115 was used for,heterologous expression,of BASI.,(2),YPD medium,(1% (w/v) yeast extract, 2% (w/v) peptone,2% (w/v) dextrose),YNB,(Yeast Nitrogen Base with ammonium sulphate without amino acids),BMSY media,2. Materials and methods,(3) To induce expression P. pastoris was grown in 500 mL BMSY media at,28C,280 rpm,.,After 24h growth, expression was induced,by adding 50% methanol,to a,concentration of 0.5%,(v/v).,To,maintain,induction 50% methanol was added to 0.5% (v/v),every 24 h,.,2. Materials and methods,2.2. Cloning,A,new polylinker,was,introduced,into,pPIC9K,by replacing the BamH INot I fragment, including the alpha mating factor secretion signal, creating pPIC9Kp (Fig. 1).,2. Materials and methods,2.3. Preparation of competent cells,(1) Conduct the step of this experiment according to the procedure of this paper.,(2)The,competent cells,were either,used right away, or,stored at 80C,for later use.,2. Materials and methods,2.4. Transformation,Prior to transformation,all plasmids,were,linearized,by BglII digestion, following,gel purification using MinElute QIAGEN,(Hilden, Germany).,(2),Transformations,were done by,electroporation,using a Gene Pulser (BIO-RAD, USA), voltage: 1500 V, capacitance: 25,F and resistance: 200,.,2. Materials and methods,2.5. Screen for single-copy transformants,(1),Single colonies,were,picked from,the,initial,YNB plates,and,resuspended,in 10L water.,(2),Cells,were,lysed,by,microwaving,for 1 min.,2. Materials and methods,2.6. Insoluble blue starch assay,(1) AMY2 (3.75 nM) was,preincubated,with,BASI,at,various,concentrations,(ranging from 0 to 50 nM) in a total volume of 400,L for 10 min at 37C in reaction buffer.,2. Materials and methods,2.7. Southern blotting,(1),Total DNA,was,purified,by using,the MasterPure,TM,Yeast DNA Purification kit,from Epicentre (Madison, USA),(2) The,quality,and,concentration,of the DNA was,determined,by,agarose gel electrophoresis,and,spectrophotometry,.,2. Materials and methods,2.8. Protein purification,(1),BASI,was,precipitated,from the,supernatant,by adding solid,ammonium sulphate (,(NH,4,),2,SO,4,) to 3.2 M final concentration,and the solution was stirred for 30 min.,(2) BASI containing fractions from the column were analysed by,SDS-PAGE,and pure fractions were pooled as the product of the purification.,Back,3. Results,3.1. Design of codon-optimized genes,(1)To investigate whether alteration of the,codon usage,in the,BASI CDR(two codon-optimized coding regions ), could,result,in higher expression levels,two,alternative,BASI genes,were,designed: one,fully codon optimized,(,arBASI,) and one where,only,arginine codons,were,altered,(,RBASI,).,3. Results,3.2. Cloning of BASI, arBASI and RBASI,(1)The original,BASI signal peptide,was,replaced,by a truncated version of the alpha mating factor secretion signal from,S. cerevisiae, in order to ensure secretion and authenticity.,(2) To enhance the initial binding of the small subunit of the ribosome to the mRNA template, the consensus,Kozak sequence,(Kozak, 1987) (,ACC,) was,inserted,just,upstream,of,the start codon,in all constructs.,3. Results,3.3. Screening for single-copy transformants,(1)In this study,single insertion transformants, generated by gene replacement of the AOX1 gene,was preferred, as,this type of transformants,is relatively easily,screened for, and multiple insertion events rarely take place.,(2) For,each transformation 100 clones,were screened, approximately,25%,of these were,single insertion transformants, generated by replacement of the AOX gene.,3. Results,(3) To,verify the exact integration site, a,Southern blot analysis,was performed, using a randomly,labelled AOX1 promoter,fragment as,probe,.,(4) As illustrated in Fig. 5, the,selected clones,were all,single insertion transformants, generated by gene replacement of the AOX1 gene, thus,making comparison,of the,expression levels,from the,various CDRs, in these particular transformants , possible.,arBASI,BASI,RBASI,pPIC9Kp empty vector,GS115,Labelled,DNA-BstEII digest was used as marker,3. Results,3.4. Expression, purification and characterization of BASI,(1)The selected single-copy AOX1 gene replacement transformants were all grown in 3 mL BMSY media at 28C.,(2),Maximum expression levels,were reached,after 4 days,for all,constructs (,Fig. 6A,).,(3) If cultures were grown for,more than 4 days,cell lysis,was observed.,Fig.6.(A),3. Results,(4)In order to,analyses,the recombinant BASI protein for the,presence,of,post-translational modifications,liquid chromatography mass spectrometry,was performed.(Domon and Aebersold, 2006 ; Mitulovic and Mechtler,2006,),(5),Activity of the recombinant protein,was assessed,using the insoluble blue starch assay.,3. Results,(6)BASI purified from P. Pastoris and from E. coli showed,identical,AMY2 inhibition,properties,(,Fig. 7,).,Back,4. Discussion,(1)During the past two decades,P. pastoris,has become a widely,used,heterologous expression system,.,(2) Apart from the level of expression, one of the,advantages,of the Pichia-based system is,secretion of the mature protein,.,4. Discussion,(3),The fact,that,the fully codon-optimized CDR,results in,lower,expression levels,than the wild-type and that,only constructs,with the alpha mating factor secretion signal,results in,secreted protein, shows that in this study ,the choice of signal,sequence,has,greater impact on,the,expression levels,than,the choice of codon usage.,4. Discussion,(4) In this study ,three genes,using different codons , but all,encoding the same,-amylase/subtilisin inhibitor from barley,have been,heterologously expressed,in P. Pastoris resulting in,65125 mg L,1,native protein.,Back,A Manual for the Preparation and Transformation of Pichia pastoris Spheroplasts,This manual is from invitrogen life technologies,tech_service,Method :,(1)Preparing the Transforming DNA and Pichia Cells,(2) Preparing Spheroplasts,(3) Transformation,A Manual for the Preparation and Transformation of Pichia pastoris Spheroplasts,Back,
展开阅读全文