1 / 13

민 연 숙

E.coli expression vector pBAD-DEST49. 민 연 숙. Description. pBAD-DEST49 is a 6.2kb vector adapted for use with the Gateway Technology. It is designed for regulated expression of N-terminal HP-thioredoxin fusion proteins in E. coli. Construct - P BAD

trang
Download Presentation

민 연 숙

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. E.coli expression vector pBAD-DEST49 민 연 숙

  2. Description • pBAD-DEST49 is a 6.2kb vector adapted for use with the Gateway Technology. It is designed for regulated expression of N-terminal HP-thioredoxin fusion proteins in E. coli. • Construct - PBAD - Ampr - pUC ori - araC

  3. Feature • His-Patch Thioredoxin (HP-thioredoxin) : 11.7kDa protein . 32 Glutamate, 64 Glutamine-> Histidine =>native 8 Histidine과 patch형성. => simple, rapid purification on metal-chelating resins • facilitate high-level expression, increase translation efficiency • EK : enterokinase recognition site : Allows removal of the N-terminal tag from the recombinant fusion protein

  4. Feature • ccdB gene : E.coli chromosome 의 degradation. • V5 epitope : Gly lys pro ile pro asn pro leu leu gly leu asp ser thr ->detection and purification • 6X His Tag : detection and purification • Two recombination sites :attR1 and attR2 > allows recombinational cloning of the gene of interest

  5. Regulation • Regulation by Arabinose • AraC protein : transcriptional regulator • Arabinose binding → release the DNA loop → allows transcription to begin

  6. Arabinose operon • Arabinose isomerase - encoded by araA • Ribulosekinase - encoded by araB • Ribulose-5-phosphate epimerase - encoded by araD CRP : promotes the rearrangement of AraC, Helps to break the DNA loop and also helps AraC to bind to araI

  7. Regulation by Arabinose 210bp Pc : araC promoter CAP : CAP(cAMP activator protein) binds to activate transcription from PBAD and Pc The CAP-cAMP complex binds to the DNA and stimulates binding of AraC to I1 and I2. cAMP activator protein(CAP)-cAMP complex binds to the DNA and stimulates binding of AraC to I1 and I2

  8. arabinose induction GFP

  9. Advantages & Conclusion • Regulated by Arabinose • Dose-dependent regulation • High protein yields • Simplified detection and purification of expressed protein

  10. The End

  11. recombination • Two recombination sites, attR1 and attR2 : allows recombinational cloning of the gene of interest

  12. ccdB gene : The ccdB gene product is toxic to all standard coli strains, thereby eliminating all coli that have been transformed with non-reacted destination vector. The ccdB protein interferes with the “ligation activity” of DNA gyrase, resulting in the degradation of chromosomal DNA and subsequent cell death.

  13. ccdB Gene Gateway technology L1 L2 Entry Clone R1 R2 Kmr Destination Vector LR Clonase Cmr (mix of 3 recombination proteins (Int, IHF & Xis) Gateway Technology : rapid and highly efficient way to move DNA sequences into multiple vector systems for functional analysis and protein expression

More Related