ExpressionL19usingPichiapastoris
Pichia pastoris is a methylotropic yeast used to express high amounts of protein. Secreted expression is achieved with a cleavalable faktor. To yield high expression strains it is important to select for multiple copies of the insert in the pichia genome. This is achieved with selection on high Zeocine concetration plates (2g/ml).Material :Glycerol or YPD agar plate containing Pichia pastorisYPD Medium (Ye......阅读全文
Expression－L19－using－Pichia－pastoris
Pichia pastoris is a methylotropic yeast used to express high amounts of protein. Secreted expression is achieved with a cleavalable faktor. To yield
毕赤酵母表达（pichia－pastoris－expression－）实验手册（3）
液体YPD培养基可常温保存；琼脂YPD平板在4℃可保存几个月。加入Zeocin 100ug / ml，成为YPDZ培养基，可以4℃条件下保存1～2周。2.4 YPDS + Zeocin 培养基（Yeast Extract Peptone Dextrose Medium）：yeast extract
毕赤酵母表达（pichia－pastoris－expression－）实验手册（4）
三．主要试验环节的操作3.1 酵母菌株的分离纯化接种GS115于5ml YPD液体培养基，30℃，200rpm振荡过夜，涂布 YPD平板，30℃培养48 小时，用 YNB基本培养基和含His的补充培养基作点种分离纯化，挑选在补充培养基生上生长而在基本培养基上不生长的单菌落划YPD平板，4℃保存。3.
毕赤酵母表达（pichia－pastoris－expression－）实验手册（2）
一．毕赤酵母表达常用溶液及缓冲液的配制1．1 各种母液的配制10*YNB （含有硫酸铵、无氨基酸的13.4%酵母基础氮源培养基） 4℃保存。34g酵母基础氮源培养基（无硫酸铵）+100g硫酸铵，溶于1000ml水中，过滤除菌。500*B （0.02%生物素 Biotin） 4℃保存 保存期为1年。2
毕赤酵母表达（pichia－pastoris－expression－）实验手册（1）
大肠杆菌表达系统最突出的优点是工艺简单、产量高、周期短、生产成本低。然而，许多蛋白质在翻译后，需经过翻译后的修饰加工，如磷酸化、糖基化、酰胺化及蛋白酶水解等过程才能转化成活性形式。大肠杆菌缺少上述加工机制，不适合用于表达结构复杂的蛋白质。另外，蛋白质的活性还依赖于形成正确的二硫键并折叠成高级结构，在
毕赤酵母表达（pichia－pastoris－expression－）实验手册（6）
随着PCR技术的不断发展成熟（扩增长度、保证性、产量和特异性等），质粒构建过程的大多数细胞内的DNA复制将被PCR这一细胞外的DNA复制所代替，质粒构建效率将有质的飞跃。4.4密码子的偏好性的原则酵母菌对外源基因的表达也和外源基因密码子的选用有关。了解表达系统宿主在密码子使用上的偏爱性对从翻译水平分
毕赤酵母表达（pichia－pastoris－expression－）实验手册（5）
3.5 Pichia酵母表达直接PCR鉴定重组子的方法3.5.1 模板的处理：1. 平板上的菌落长到肉眼可见时（约12小时）；2. 将除了模板之外的其它PCR反应液的组分准备好，并分装。引物最好使用Kit中已有的检测专用的引物，或者或者一条使用载体上的引物，一条使用基因的特异性引物（这样做可以鉴定非
Expression－Library－Screening－(Procaryotic)－Using－APFusion－Proteins
Outline:Bacteriophage lambda is a linear double stranded DNA, approximately 50 kB in size. The two sticky ends help the phage to recircularize after e
关于甲醇酵母表达系统的基本介绍
甲醇酵母基因表达系统是一种最近发展迅速的外源蛋白质生产系统。甲醇酵母是可利用甲醇作为唯一碳源的酵母，主要有H.Polymorpha、Candida Bodinii、Pichia Pastoris三种，其中Pichia Pastoris作为基因表达系统使用得最多、最广泛。与以往的基因表达系统相比，
木聚糖酶的参数功能
木聚糖酶是半纤维素降解的关键酶，我国用橄榄绿链霉菌基因转到毕赤酵母Pichia pastoris获得高效表达。酶活性为1 200 U/mL，比活性为2 869 U/mg。具非常好的抗蛋白酶降解能力。
重组乙型肝炎疫苗甲基营养型酵母的简介
共有4种表达系统。甲基营养型酵母生产HbsAg具有培养基廉价，目的基因与酵母染色整合的优点， 不仅可克服质粒丢失，还可提高表达量。汉逊酵母（Hansenula Polymorpha，H.P.）和毕赤酵母（Pichia Pastoris，P.P.）系统已应用于工业化生产。已上市的酿酒酵母和甲基营养
毕赤酵母如何做表达
Pichia.pastoris酵母菌体内无天然质粒，所以表达载体需与宿主染色体发生同源重组，将外源基因表达框架整合于染色体中以实现外源基因的表达。包括启动子、外源基因克隆位点、终止序列、筛选标记等。表达载体都是穿梭质粒，先在大肠杆菌复制扩增，然后被导入宿主酵母细胞。为使产物分泌胞外，表达载体还需带有
简述甲醇酵母表达系统的应用前景
经过近几年的发展完善，甲醇酵母表达系统已日趋成熟，应用也日趋广泛。国内已有多篇在甲醇酵母中生产外源蛋白质成功的报道。美国Invitrogen公司也已开发出多种新型的甲醇酵母表达系统试剂盒，如Multi-copy Pichia Expression Kit、Easyselect Pichia Ex
Using－GenBank
GenBank(R) is a comprehensive database of publicly available DNA sequences for more than 205,000 named organisms and for more than 60,000 within t
Using－a－Counting－Chamber
Using a Counting ChamberFor microbiology, cell culture, and many applications that require use of suspensions of cells it is necessary to determine ce
Pichia酵母表达系统操作方法
我先说我有的质粒和菌株：PPIC9K，PPIC9，PPICZahphaA，B，C以及一个改造的PPIC9K载体，EcoRI和BamHI双酶切，C末端引入了一个6histag菌株：GS115，KM71，X33，以及十几个空载体对照菌株我的酵母表达Protocol：>10ug质粒用Sal线性化，加1/1
Manipulating－Expression－of－Tonoplast－Transporters
Plant vacuoles have multifaceted roles including turgor maintenance, cytosolic pH and ionic homeostasis, plant protection against environmental st
Protein－Expression－and－Purification－Protocol
Step 1: Transform appropriate DNA plasmid into BL21(DE3) E. coli cells. These cells must be competent. (Protocol for how to make competent cells.)a) T
Deglycosylation－of－Glycoproteins－Using－Endoglycosidases
T.H. Plummer, Jr. and A.L. Tarentino, Department of Biochemistry, New York State Department of Health, Wadsworth Center for Laboratories and Research,
ChIP－using－plant－samples
实验概要 The immunoprecipitation (IP) of cross-linked chromatin with antibodies specific for certain histone modifications (chromatin immunoprecipitati
Measurement－of－Green－Fluorescent－Protein－Expression
ReagentsCells to be studied expressing green fluorescent protein (GFP). Note that the same cell type without GFP is needed as control.Hoechst 33342 s
FOSB－gene－expression－and－drug－abuse
Drug addiction is associated with long-term behavioral changes, suggesting a long-lived transcriptional regulator that responds to chronic drug exposu
Transfection－of－Mammalian－Cells－Using－Lipofectamine
Materials: LipofectamineBasal Medium containing 10% fetal bovine serum, 1% glutamine, 1% aaBasal Medium containing 1% glutamineBasal Medium cont
Method:－Cell－Counts－Using－a－Hemacytometer
Method: Cell Counts Using a HemacytometerJune 1, 1990Rosalie VeilePurpose:The purpose of this procedure is to determine the cell density of the cultur
Using－GenBank－for－Genomic－Authentication:－A－Tutorial
The GenBank database is perhaps one of the most important repositories of genetic information. A researcher working in the field of genomic authe
ChIP－using－plant－samples－–－Arabidopsis
实验概要This protocol describes how chromatin is prepared from Arabidopsis, which can subsequently be used for chromatin immunoprecipitation (ChIP). T
Extraction－of－DNA－using－DNAzol®－Reagent
实验概要DNAzol® Reagent (Genomic DNA Isolation Reagent) is a complete and ready-to-use reagent for the isolation of genomic DNA from solid and liquid sa
Fluorescence－In－Situ－Hybridization－using－TSA™
实验概要This protocol describes steps for fluorescent in situ hybridization (FISH) to Drosophila embryos using Tyramide Signal Amplification (TSA™), and
RNA－extraction－using－trizol/tri
RNA extraction with TRIzol (Invitrogen product name) or the equivalent TRI (Sigma-Aldrich product name) is a common method of total RNA extraction fro
MicroRNA－Expression－Profiling－by－Bead－Array－3
Table 2 Modulation of microRNA expression by IFNα—4 and 24 h after stimulation ControlInterferon-alpha treated4 h24 h4 h24 haME-15HuH7CHFME-15HuH7CHFM