酵母表达系统
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Yeast Expression System Lihong Xu, PhD
Technical support
Invitrogen corporation
Hotline:8008208181-2-2
Email:cntechsupport@
Invitrogen Protein Expression Systems
-Prokaryotic: E. coli
-Cell-free (and Lumio technology)
-Yeast: Pichia Pastoris
-Insect: Baculovirus
-Mammalian
-Mammalian Viral
Expression hosts and their applications
酵母表达系统的优缺点
Invitrogen ’s Yeast Expression Systems
•Bioproduction
•Industrial •
Not as well known as P. pastoris •High-level expression •Amenable to large-scale bioproduction
•Licensing for industrial is less restrictive
Pichia methanolica •E. coli user who
wants to move to a eukaryote
•Expression not as high as Pichia •Typically uses nutritional markers
rather than antibiotics
•Very commonly used –mostly for genetic studies •Good alternative to E. coli if eukaryote needed •Variety of vectors available Sacchromyces cerevisiae •Need lots of
protein
•Bioproduction
•Requires license by industrial •High-level expression •Amenable to large-scale bioproduction •Thousands of publications about the system
•Wide range of products
Pichia pastoris Customer ’s
needs
Disadvantage Advantage System
Yeast Expression Systems
Pichia Pastoris •High level of protein expression.
•Many vector choices available, some with antibiotic selection.
•No Gateway or TOPO adapted vectors available.
•Can obtain multi-copy integrants in the chromosome for high expression.
•Constitutive and inducible promoters available.
•Protein can be expressed intracellularly or secreted with current vectors.
•Licensing required primarily for industrial customers.
•High biomass –the cell density of Pichia pastoris can be 10 times
greater than that of Saccharomyces cerevisiae
•Strong promoters –both inducible(AOX1) and constitutive promoters(GAP) are available to drive high-level expression -Typically, 30% of the total soluble protein in methanol induced cells is alcohol oxidase -Constitutive expression can be achieved using the promoter from the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene
•Efficient secretion –Recombinant proteins fused to the α-factor or a native secretion signal will be directed into the medium, simplifying purification
Three key factors to achieve high yields
S. Cerevisiae P. pastoris
Overview: The Pichia Advantage •A simple, convenient method for rapidly preparing and transforming competent Pichia pastoris cells eliminating the tedious and time-consuming preparation of spheroplasts
•Vectors containing the Zeocin™or Blasticidin resistance gene to allow direct selection of transformed cells. No more hassling with drop-out medium or screening through background colonies
•The growth medium does not require growth factors or supplements which are costly and make protein purification more difficult
•In addition, vectors have been designed with fusion tags that simplify the purification and analysis of expressed proteins.
Pichia advantage-proven expression
Multi-Copy Pichia Expression Kit(K175001)
EasySelect Pichia expression kit (K174001)
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Flowchart of expression in Pichia Pastoris(Easy-select)
Pichia pastoris expression vextors
Inducible expression system with Zeocin selection Constitutive expression system
with Zeocin selection
Inducible expression system
with Blasticidin selection
Multi-copy Pichia expression kit
Flowchart of expression in Pichia Pastoris(Multicopy)
Integration into Pichia
Genome
His+ MutS
In vitro Multimerization. Excise expression cassette BglII/ BamH1Ligate cassettes to form multimers Ligate multimers into vector BglII/BamH1
Insertion Replacement Multiple Insertions
Signal cleveage
Pichia Pastoris Strains
Pichia Pastoris expression kits components
Pichia Methanolica •High level of protein expression.
•Only two vectors offered, no antibiotic selection.
•No Gateway or TOPO adapted vectors available.
•Cannot be used to select for multi-copy integrants.
•Only inducible promoter available.
•Protein can be expressed intracellularly or secreted with current vectors.
•Less licensing requirements than Pichia pastoris(industrial customers that cannot use Pichia pastoris can be suggested to use this system instead).
Pichia methanolica
pMET and pMETα
•Commercial alternative for those where
Pichia Pastoris licensing issues are too
restrictive.
•AUG1 and AUG2 instead of AOX1, AOX2. •Auxotrophic selection only
Strains
•Lower level of protein expression than Pichia due to lower biomass accumulation.
•Many vector choices available for low and high copy, some with antibiotic selection.
•Gateway and TOPO adapted vectors are available.
•Vectors do not integrate into the chromosome.
•Only inducible promoter available.
•Protein can only be expressed intracellularly with current vectors.•Less licensing requirements than Pichia pastoris.
IN VS c1, C810-00, is a fast-growing diploid strain ideal for expression.
Sc Vectors: summary
YES™Vector Collection
Description Origin Selection Marker Tag
pYES2/NT 2 µURA3NT Xpress™
pYES2/CT 2 µURA3CT
V5-6xHis
pYES2.1/V5-HIS-TOPO* 2 µURA3CT
V5-6xHis
pYES3/CT 2 µTRP1CT
V5-6xHis
pYES6/CT 2 µBlasticidin CT
V5-6xHis
pYC2/NT CEN6/
ARSH4
URA3
NT
Xpress™
pYC2/CT CEN6/
ARSH4
URA3
CT
V5-6xHis
pYC6/CT CEN6/
ARSH4
Blasticidin
CT
V5-6xHis
Now available product for Sc expression system •Saccharomyces cerevisiae System Kits
•pYES2.1 TOPO TA Expression Kit K415001 K4150-01
•pYES2-DEST52 Gateway Dest. Vect. 12286-019 12286019
•pTEF1/Bsd Starter Kit K51301 K513-01
•S.c. EasyComp Transformation Kit K505001 K5050-01
•Saccharomyces Strains
INVSc1 C81000 C810-00
•Saccharomyces Vectors (Sold Individually)
•pTEF1/ZEO V50320 V503-20
•pTEF1/Bsd V51320 V513-20
•pYES2 V82520 V825-20
•pYES2/CT V825120 V8251-20
•pYES2/NT A, B, C V825220 V8252-20
•pYES2-DEST52 Gateway Dest. 12286-019 12286019
•pYES3/CT V825320 V8253-20
•pYC2/CT V825520 V8255-20
•REAGENTS REQUIRED TO MAKE YPAD MEDIA
•30391023 Select Agar
毕赤酵母系统表达常见问题及可能的原因
•克隆过程:
读码框是否正确(tag/信号肽序列同框)
胞内表达要有ATG
密码子偏性问题
不能有提前终止序列(TTTTTATA)
PCR/Southern blot鉴定GOI整合
•表达环节:
同时筛选Mut+和Mut S
筛选多个克隆进行表达鉴定(最少6-10个克隆);
表达时间曲线,最适收样时间
分泌表达:同时检测上清和胞内;上清用硫酸铵沉淀或超滤浓缩后再检测;
胞内表达:细胞破壁情况、蛋白降解问题等
是否用WB或其它功能活性检测等更敏感的方法检测
WB:转膜/抗体合适
•小规模能检测到蛋白表达,放大规模培养后未检测到表达(阳性对照有表达)
-重新进行条件优化
-选择高拷贝整合菌株进行表达;
-蛋白降解问题:用unbuffered media
加1% Casamino acids to buffered media -同时尝试胞内和分泌表达系统
-用自身分泌信号分泌表达
•Pre-pro-insulin:
Currently recombinant insulin is made in E. coli, the two different chains in separate E. coli strains with final assembly of the two
chains in vitro. It’s been tried in S. cerevisiae and didn’t work.
Insulin processing requires the removal of an N-terminal signal AND an internal portion, followed by proper di-sulfide bond formation linking the two chains.
Thank you!。