1 / 46

Novel Strategies for Expression of High Purity, Soluble, Active Proteins from E. coli

rusk
Télécharger la présentation

Novel Strategies for Expression of High Purity, Soluble, Active Proteins from E. coli

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. 1

    2. 2

    3. 3 Outline Cold Shock Technology pCold Expression Vectors pCold TF Chaperone Plasmid Set Single Protein Production System SPP System Purification Of His-Tagged Proteins

    4. 4 What is Cold Shock Technology? The system induces protein expression at low temperatures (15OC), suppressing synthesis of most other host proteins. Results in up to 60% of all the expressed cellular protein as target protein. Reduces potentially destructive cellular protease activity. Lower temperature increases solubility

    5. 5 Takaras pCold Expression Vectors Uses selective elements from cspA gene cspA promoter , 5 codon gene cspA 5UTR cspA 3 end transcription terminator site Tags and MCS His Tag TEE (Translation Enhancing Element) Factor Xa

    6. 6 Strategy of cold shock vector development

    7. 7

    8. 8 What is the Cold Shock Technology expression method?

    9. 9

    10. 10

    11. 11 Expression level and Solubility increased vs T7

    12. 12 Comparative study with pulse-labeling experiment vs T7

    13. 13 Purification of expressed protein (SDS-PAGE)

    14. 14 High-throughput protein expression in 96-well format

    15. 15

    16. 16 What are the benefits of cold shock protein expression? High purity- high yield recombinant protein expression Low temperature protein induction (15OC) Reduces host protease activity Increases solubility / folding Expresses difficult proteins High Throughput Compatible with Takaras Chaperone Plasmid Vectors

    17. 17 Outline Cold Shock Technology pCold Expression Vectors pCold TF Chaperone Plasmid Set Single Protein Production System SPP System Purification Of His-Tagged Proteins

    18. 18 Chaperone Plasmid Set Family of heat shock & related proteins called molecular chaperones as they facilitate movements and correct folding of polypeptides in cells 5 different plasmids, each of which is designed to express multiple molecular chaperones Co-transformation & expression of a target protein with one of these chaperone teams increases the recovery of soluble proteins in E.coli

    19. 19

    20. 20

    21. 21 In vitro Folding Products Refolding CA Kit (code 7350) Cycloamylose (CA), an artificial chaperone, for surfactant removal and recovery of protein activity. An easy 2-step procedure for optimizing the refolding conditions o f inclusion body proteins. Chaperonin GroE (code 7330) Takara GroE can be used for refolding denatured proteins to recover functional activity. Corystein (purothionin) (code 7311) Protein refolding through exchange reactions between disulfide bonds

    22. 22 Outline Cold Shock Technology pCold Expression Vectors pCold TF Chaperone Plasmid Set Single Protein Production System SPP System Purification Of His-Tagged Proteins

    23. 23 pCold TF (Trigger Factor) Takara's pCold TF DNA Vector is a fusion of cold shock expression vector technology and expression of Trigger Factor (TF) chaperone as a soluble tag. TF is a prokaryotic ribosome-associate chaperone protein (48 kDa). The pCold TF DNA Vector = high yield protein expression + chaperone expression to facilitate correct protein folding No requirement for Chaperone plasmids

    24. 24

    25. 25

    26. 26 What are the benefits of pCold TF expression? All the benefits of pCold Vectors plus TF chaperone allows for proper protein folding and soluble protein expression Easy to use

    27. 27 Summary

    28. 28 Outline Cold Shock Technology pCold Expression Vectors pCold TF Chaperone Plasmid Set Single Protein Production System SPP System Purification Of His-Tagged Proteins

    29. 29 MazF E. coli toxin classified gene Possesses mRNA endoribonuclease activity and is very similar in enzymatic function to RNAse A Specifically cleaves single-stranded cellular RNA at the 5end of ACA sequences Does not cleave double-stranded RNA nor does it cleave single or double-stranded DNA

    30. 30 Addiction Modules (MazE/MazF) MazE/MazF????? ????????????????????MazF????????MazE???????????????????????????? ???????????????????????????????ClpA?????????????MazE?ClpA???????????????????????MazF???????????????????mRNA?????????????????????????MazE/MazF????? ????????????????????MazF????????MazE???????????????????????????? ???????????????????????????????ClpA?????????????MazE?ClpA???????????????????????MazF???????????????????mRNA?????????????????????????

    31. 31 What is the SPP System (Single Protein Production System)? E. coli based two vector co-expression system Utilize modified pCold Vectors Vector carrying endoribonuclease MazF gene

    32. 32 pCold (SP4) vectors Modified pCold vectors to contain no ACA sequences (i.e ACA-less) Retains all of the benefits of the pCold vector technology SPP System pCold Vectors

    33. 33 Single Protein Production System SPP SystemTM SPP???? Single Protein Production System(SPP SystemTM)?MazF??????????????????????????? ??????????????????????????????(????)???????????????????????????? MazF??????????????ACA??????????????????????????2?????????????? ?????????????mRNA?ACA?????MazF???????????????????????????? ???ACA?????????????mRNA??????????????????????? SPP????????????????????????????????????????? ????:Molecular Cell, Vol. 18, 253-261, 2005SPP???? Single Protein Production System(SPP SystemTM)?MazF??????????????????????????? ??????????????????????????????(????)???????????????????????????? MazF??????????????ACA??????????????????????????2?????????????? ?????????????mRNA?ACA?????MazF???????????????????????????? ???ACA?????????????mRNA??????????????????????? SPP????????????????????????????????????????? ????:Molecular Cell, Vol. 18, 253-261, 2005

    34. 34 Single Protein Production PP SystemTM SPP Sysytem??????????????? ??????????????????????SPP System?????????????? SPP System???????????????????????? ???????TEE???His-Tag???FactorXa????????4????????????????SPP Sysytem??????????????? ??????????????????????SPP System?????????????? SPP System???????????????????????? ???????TEE???His-Tag???FactorXa????????4????????????????

    35. 35 Single Protein Production System SPP SystemTM MazF???????? SPP System??????MazF??????????? pCold(SP4)??????????????????????????????MazF???????? SPP System??????MazF??????????? pCold(SP4)??????????????????????????????

    36. 36 ????????????? ????????????????(No Insert)????????????MazF???????????(MazF-)????????????????????MazF???????(MazF+)?????????????????? ???MazF?EnvZB??????????????????????????EnZB(?)?????????????? MazF??????????????????????????????????????? ????????????? ????????????????(No Insert)????????????MazF???????????(MazF-)????????????????????MazF???????(MazF+)?????????????????? ???MazF?EnvZB??????????????????????????EnZB(?)?????????????? MazF???????????????????????????????????????

    37. 37 ACA?????????? ??????:MazF???????????(MazF-)?????????????Trx???????????????????????????MazF???????(MazF+)?????????????????????????????ACA?????????Trx???(ACA+)??Trx?mRNA?MazF???????????????????ACA??????Trx???(ACA-)???Trx?????????????????????????????? CBB??:ACA??????????(ACA-)?MazF???????(MazF+)Trx????????????????? ACA??????????????????????????????ACA?????????? ??????:MazF???????????(MazF-)?????????????Trx???????????????????????????MazF???????(MazF+)?????????????????????????????ACA?????????Trx???(ACA+)??Trx?mRNA?MazF???????????????????ACA??????Trx???(ACA-)???Trx?????????????????????????????? CBB??:ACA??????????(ACA-)?MazF???????(MazF+)Trx????????????????? ACA??????????????????????????????

    38. 38 ?????????????????? ??Eotaxin?????????????????????? MazF????(MazF-)???????????????Eotaxin(?)??????????????(????????)???????? MazF???????(MazF+)???3??????????????????????72???????????????????????????????????? ??Eotaxin?????????????????????? MazF????(MazF-)???????????????Eotaxin(?)??????????????(????????)???????? MazF???????(MazF+)???3??????????????????????72??????????????????

    39. 39 Exceptional Signal to Noise Ratio Increased Protein Stability and Solubility Excellent for labeling applications (i.e.NMR) What are the benefits of the SPP System (Single Protein Production)?

    40. 40 Cold shock technology

    41. 41

    42. 42 Outline Cold Shock Technology pCold Expression Vectors pCold TF Chaperone Plasmid Set Single Protein Production System SPP System Purification Of His-Tagged Proteins

    43. 43 immobilized metal affinity chromatography (IMAC) resin utilizing Co2 strict requirements for spatial positioning of only adjacent / spacial histidines. Nickel-based resins - spatial requirements less strict. Uniform Matrix. All reactive sites in TALON Resin look like three-dimensional pockets. Less background (esp. E.coli SlyD common contaminant)

    44. 44

    45. 45

    46. 46

    47. 47

More Related