T7 RNA Polymerase, His-tagged
Cat No. FNK-RP-T7RP-S
Quantity: 500KU
Description
T7 RNA Polymerase is a widely used enzyme in molecular biology, and the His-tagged version offers several advantages for purification and use. Here's a concise overview of His-tagged T7 RNA Polymerase:
1. Purification: His-tagged T7 RNA Polymerase can be purified using a single-step metal-affinity chromatography method. This technique yields large amounts of highly active polymerase with approximately 97% homogeneity[5].
2. Expression: The enzyme is typically expressed in E. coli systems. Different strains like BL21, SG13009, and XL1-Blue have been used for expression, with XL1-Blue showing the best results for producing soluble protein[5].
3. Optimization: The yield of soluble, His-tagged T7 RNA Polymerase can be optimized by adjusting growth conditions such as temperature, inducer concentration (IPTG), and the presence of organic osmolytes like sorbitol and glycine betaine[5].
4. Function: His-tagged T7 RNA Polymerase is a DNA-dependent RNA polymerase that specifically recognizes T7 phage promoters. It's designed to function at higher temperatures than the wild-type enzyme, with an optimal temperature range of 50-52°C[2].
5. Applications: This enzyme is crucial for in vitro RNA transcription and is used in various molecular biology techniques. The His-tag allows for easy purification without compromising the enzyme's activity[1][3].
6. Purification protocol: A typical purification process involves cell lysis, followed by loading the lysate onto a nickel-charged column. The protein is then eluted using an imidazole-containing buffer[4].
The His-tagged version of T7 RNA Polymerase combines the high specificity and efficiency of the original enzyme with the convenience of easy purification, making it a valuable tool in molecular biology research and applications.
Citations:
[1] https://pubmed.ncbi.nlm.nih.gov/9591113/
[2] https://www.neb.com/en-us/products/m0658-hi-t7-rna-polymerase
[3] https://www.tandfonline.com/doi/abs/10.2144/98245bm03
[4] https://williams.chemistry.gatech.edu/course_Information/4581/labs/prot_pur/prot_pur.html
[5] https://pubmed.ncbi.nlm.nih.gov/15642476/
