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Invitrogen™
Champion™ pET SUMO Expression System
The Champion™ pET SUMO Expression System produces the highest levels of soluble protein in E. coli. It utilizes a small深入閱讀
| 產品號碼 | Quantity |
|---|---|
| K30001 | 20 reactions |
產品號碼 K30001
價格 (HKD)
8,975.00
Each
Quantity:
20 reactions
Delivery information
Standard Products - 1-2 business days for Beijing, Shanghai, Guangzhou, Shenzhen and provincial capitals, 2-3 business days for 2nd-tier cities, and 4 business days for 3rd-tier cities and remote areas.
Air Freight Restricted Products - 2-4 business days for Beijing, Shanghai, Guangzhou, Shenzhen and provincial capitals, 3-5 business days for 2nd-tier cities, and 6-10 business days for 3rd-tier cities and remote areas.
Supply Center: Delivered from the Supply Center closest to you.
Air Freight Restricted Products - 2-4 business days for Beijing, Shanghai, Guangzhou, Shenzhen and provincial capitals, 3-5 business days for 2nd-tier cities, and 6-10 business days for 3rd-tier cities and remote areas.
Supply Center: Delivered from the Supply Center closest to you.
價格 (HKD)
8,975.00
Each
The Champion™ pET SUMO Expression System produces the highest levels of soluble protein in E. coli. It utilizes a small ubiquitin-related modifier (SUMO) fusion, belonging to the growing family of ubiquitin-related proteins, to enhance the solubility of expressed fusion proteins. In contrast to ubiquitin, SUMO is involved in the stabilization and localization of proteins in vivo. After expression, the 11 kd SUMO moiety can be cleaved by the highly specific and active SUMO (ULP-1) protease at the carboxyl terminal, producing a native protein*. The Champion™ pET SUMO Protein and Peptide Expression System offers:
• Greatly enhanced solubility with an N-terminal SUMO fusionHighly efficient cleavage- produces native protein of interest with SUMO (ULP-1) protease*Highly specific cleavage- eliminates the chance of your protein of interest being internally digested, regardless of its amino acid sequenceSignificantly increased stability with SUMO fusion-can be used for small peptide productionT7lac promoter for high-level protein expressionN-terminal 6xHis tag for protein detection and purification
• Greatly enhanced solubility with an N-terminal SUMO fusionHighly efficient cleavage- produces native protein of interest with SUMO (ULP-1) protease*Highly specific cleavage- eliminates the chance of your protein of interest being internally digested, regardless of its amino acid sequenceSignificantly increased stability with SUMO fusion-can be used for small peptide productionT7lac promoter for high-level protein expressionN-terminal 6xHis tag for protein detection and purification
For Research Use Only. Not for use in diagnostic procedures.
規格
Antibiotic Resistance BacterialKanamycin (KanR)
Bacterial or Yeast StrainBL21(DE3)
CleavageSUMO
Constitutive or Inducible SystemInducible
Expression MechanismCell-Based Expression
Expression SystemE. coli
Inducing AgentIPTG
Product TypeExpression System
Quantity20 reactions
Selection Agent (Eukaryotic)None
VectorpET
Cloning MethodTOPO
Product LineChampion™, TA Cloning™
PromoterT7, lacO
Protein TagHis Tag (6x), SUMO Tag
Unit SizeEach
內容物與存放
The Champion™ pET SUMO Expression System is provided as a complete system. The Champion™ pET SUMO TA Cloning™ box contains linearized Champion™ pET SUMO vector, sterile water, dNTPs, 10X PCR Buffer, control template and primers, T4 DNA ligase, 10X ligation buffer, primers for sequencing or PCR screening, and an expression control. Store at -20°C. The SUMO Protease box contains SUMO Protease and buffers. Store at -80°C. The One Shot™ TOP10 box contains twenty-one 50 μl aliquots of chemically competent E. coli, S.O.C. medium, and a control plasmid. Store at -80°C. The One Shot¤ BL21(DE3) box contains twenty-one 50 μl aliquots of chemically competent E. coli, S.O.C. medium, and a control plasmid. Store at -80°C.
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Product Information
常見問答集 (常見問題)
有多种措施可防止由毒性基因本底水平表达带来的问题。这些方法均基于T7或Champion表达质粒的设计和构建是正确的：
•在不含T7 RNA聚合酶（即，DH5α）的菌株中繁殖和维持表达质粒。
•如果使用BL21 (DE3)菌株，应在室温而非37°C下生长24-48小时。
•新鲜转化严格调控的大肠杆菌菌株，如BL21-AI菌株。
•转化实验后，将转化反应接种在含100 μg/mL氨苄青霉素和0.1%葡萄糖的LB板上。葡萄糖可抑制T7 RNA聚合酶的本底表达。
•完成BL21-AI菌株的转化后，挑选3或4个转化株直接接种到新制备的含100 μg/mL 氨苄青霉素或50 μg/mL 羧苄青霉素（以及0.1%葡萄糖，如果需要的话）的LB培养基中。当培养物的OD600值达到0.4时，可加入终浓度为0.2%的L-阿拉伯糖来诱导重组蛋白的表达。
•在表达实验中，在生长培养基中补充0.1%葡萄糖和0.2%阿拉伯糖。
•尝试使用调控型细菌表达系统，如我们的pBAD系统。
通常，如果您看见1-2条主带，则表示密码子使用偏好导致了翻译过早终止。发生降解时，通常可看到梯度条带。发生降解时，可尝试在裂解缓冲液中加入蛋白酶抑制剂有助于防止降解。如果是这种情况，可通过时间点实验来确定收细胞的最佳时间。
如果存在溶解性问题，可尝试在诱导时降低温度或减少IPTG用量。也可尝试使用不同的、更严格的菌株进行表达。此外，在表达期间，也可将细菌培养基的葡萄糖含量增加至1%。
使用新鲜的细菌培养物进行接种，因为新鲜的细菌菌落通常可得到较高的蛋白得率。
检查重组蛋白序列中的不常用密码子。利用常用密码子代替稀有密码子，可显著提高表达水平。例如，精氨酸密码子AGG和AGA是大肠杆菌的不常用的密码子，所以这些密码子的tRNA水平低。
进行蛋白纯化时，在缓冲液中加入蛋白酶抑制剂，如PMSF。使用新鲜配制的PMSF，因为PMSF被稀释到水溶液中后30分钟内会失效。
如果您在表达实验中使用氨苄青霉素进行筛选，则可能会因为筛选条件缺失而出现质粒不稳定。这是因为氨苄青霉素被β-内酰胺酶破坏，或者在由细菌代谢物造成的酸性培养基条件下发生水解。在转化和表达实验中，可使用羧苄青霉素替代氨苄青霉素。
重组蛋白可能对细菌细胞具有毒性作用。应选择更严格调控的系统用于表达，如BL21-AI。也可考虑尝试使用不同的表达系统，如pBAD系统。
当您的目的基因具有毒性时这种情况很常见。尝试使用更严格调控的系统，如BL21 (DE3) (pLysS)或BL21 (DE3) (pLysE)或BL21(AI)。
引用資料與參考文獻 (1)
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引用資料與參考文獻
Abstract
A novel hematopoietic granulin induces proliferation of goldfish (Carassius auratus L.) macrophages.
Journal:J Biol Chem
PubMed ID:16473876
'Granulins are a group of highly conserved growth factors that have been described from a variety of organisms spanning the metazoa. In this study, goldfish granulin was one of the most commonly identified transcripts in the differential cross-screening of macrophage cDNA libraries and was preferentially expressed in proliferating macrophages. Unlike
1 total citations