trc Expression System ( pTrcHis TOPO TA Expression System ) 약학대학 2005-30580 조승주 - PowerPoint PPT Presentation

slide1 n.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
trc Expression System ( pTrcHis TOPO TA Expression System ) 약학대학 2005-30580 조승주 PowerPoint Presentation
Download Presentation
trc Expression System ( pTrcHis TOPO TA Expression System ) 약학대학 2005-30580 조승주

play fullscreen
1 / 11
trc Expression System ( pTrcHis TOPO TA Expression System ) 약학대학 2005-30580 조승주
334 Views
Download Presentation
marc
Download Presentation

trc Expression System ( pTrcHis TOPO TA Expression System ) 약학대학 2005-30580 조승주

- - - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

  1. < Prokaryotic Expression > trc Expression System (pTrcHis TOPO TA Expression System) 약학대학 2005-30580 조승주

  2. Prokaryotic Expression Systems -Invitrogen-

  3. trc Expression System trc promoter → The trc promoter, a hybrid promoter containing the -35 region from the trpB promoter and the -10 region from the lacUV5 promoter for high-level expression in E. coli. → Inducible promoter → Repressed by the lac repressor (lacI) → Chemically induced with iospropyl-β-D-thiogalactopyranoside (IPTG)

  4. pTrcHis TOPO TA Expression System –Invitrogen-

  5. Description of the Vectors → The trc promoter → The lacO sequence for binding the Lac repressor encoded by the lacIq gene. In the absence of IPTG, Lac repressor binds to the lacO sequence, repressing transcription. Upon addition of IPTG, expression is induced. → rrnB antitermination sequence the reduces premature transcription termination. → T7 gene 10 translational enhancer sequence for more efficient translational initiation. → A minicistron containing nucleotides that are efficiently translated in prokaryotic cells for enhanced translational efficiency. → Xpress epitope is part of the bacteriophage T7 gene 10 protein

  6. pTrcHis2 TOPO Cloning Site

  7. TOPO Cloning → Single 3’-thymidine (T) overhangs for TA Cloning → Topoisomerase I covlently bound to the vector (this is referred to as “activated” vector)

  8. Experimental Outline

  9. Features of pTrcHis TOPO

  10. Features of pTrcHis TOPO

  11. pTrcHis-TOPO TA Expression → Efficient cloning of PCR products into a prokaryotic expression vector for high-level → Regulated expression from the trc promoter → Minicistron element for enhanced translation efficiency of eukaryotic proteins in E. coli. → Provided linearized and activated with topoisomerase I for 5-minute TOPO Cloning with >85% cloning efficiency → Symplify protein purification and detection N-terminal Xpress epitope for detection with an Anti-Xpress Antibody N-terminal polyhistidine (6xHis) tag for purification using nickel-chelating resin and detection with an Anti-HisG Antibody Enterokinase cleavage site for efficient removal of N-terminal fusion tags