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Recombinant protein expression in E.coli
Recombinant protein expression in E.coli
Recombinant protein expression in E.coli
Recombinant protein expression in E.coli
Recombinant protein expression in E.coli
Recombinant protein expression in E.coli
Recombinant protein expression in E.coli
Recombinant protein expression in E.coli
Recombinant protein expression in E.coli
Recombinant protein expression in E.coli
Recombinant protein expression in E.coli
Recombinant protein expression in E.coli
Recombinant protein expression in E.coli
Recombinant protein expression in E.coli
Recombinant protein expression in E.coli
Recombinant protein expression in E.coli
Recombinant protein expression in E.coli
Recombinant protein expression in E.coli
Recombinant protein expression in E.coli
Recombinant protein expression in E.coli
Recombinant protein expression in E.coli
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Recombinant protein expression in E.coli

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Recombinant Protein expression in E.coli, Best suitable strains for protein expression, advantages of using E.coli for choosing the host for protein expression

Recombinant Protein expression in E.coli, Best suitable strains for protein expression, advantages of using E.coli for choosing the host for protein expression

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  1. Recombinant Protein Expression in E.coli Bio-Resource www.technologyinscience.blogspot.com
  2. Protein Expression in E.coli • Procaryotic systems are well studied and widely used for protein expression www.technologyinscience.blogspot.com
  3. Advantages of E.coli System • Simple, well-understood genetics. • Its very easy to genetically manipulate. • Culturing cost is minimal. • Expression is fast, since the doubling time is only 20 - 30 mins. • Well established labeling protocols for stability studies. • Established regulatory track record. • Fermentation, easy to scale up. • Inclusion bodies may be easy to purify. www.technologyinscience.blogspot.com
  4. Protein Expression in E.coli • Intra Cellular via Inclusion Bodies • Expression Yield: 1 – 5 grams of Protein / Litre • Examples: Insulin www.technologyinscience.blogspot.com
  5. E.Coli Strain & Application • XLI-Blue, DH5-alpha, top10 : Routine Cloning/Sub-cloning, Blue/white screening • XL10-Gold, MegaX DH10b : Very high efficiency cloning e.g. for library construction • XL1-Blue MR : Cloning of unmethylated DNA • JM110, INV110 : Production of unmethylated DNA • Sure, Stbl4 : Cloning of unstable plasmids • BL21 (DE3) : Expression from T7 promoter • BL21 (DE3) pLysS : Expression from T7 promoter, tight regulation • BL21 codon plus, Rosetta : Expression from T7 promoter with codon bias correction • Origami : Improved disulphide bond formation • Mach1 : Fast cloning (due to quick cell growth) www.technologyinscience.blogspot.com
  6. Vectors • Vehicle / Carrier of Foreign DNA Types of Vectors • Plasmids, • Viruses, • Cosmids, and • Artificial chromosomes www.technologyinscience.blogspot.com
  7. Vectors for Protein Expression & Features • Selectable marker • Regulatory gene (repressor) • Origin of replication • Promoter • Transcription terminator • Shine-Delgarno sequence • Start codon and Stop codon • Tags and fusion proteins • Protease cleavage site • Multiple cloning site www.technologyinscience.blogspot.com
  8. pUC19 Vector www.technologyinscience.blogspot.com
  9. Most widely used vectors • pET series from Novagen • pALTER from Promega • pCal-n from Stratagene • pBAD/His from Invitrogen, etc www.technologyinscience.blogspot.com
  10. Cloning & Screening www.technologyinscience.blogspot.com
  11. Plasmid Extraction / Isolation www.technologyinscience.blogspot.com Qiagen Plasmid Extraction Kit Plasmid Extraction Procedure
  12. Restriction Digestion & Ligation www.technologyinscience.blogspot.com
  13. Restriction Digestion and Gel www.technologyinscience.blogspot.com
  14. Transformation and Expression www.technologyinscience.blogspot.com
  15. Transformation - Procedure www.technologyinscience.blogspot.com
  16. Shake Flask Culture- Protein Expression & Analysis www.technologyinscience.blogspot.com
  17. Lac Operon & IPTG Induction www.technologyinscience.blogspot.com
  18. Protein Analysis – SDS PAGE www.technologyinscience.blogspot.com
  19. Protein Characterization www.technologyinscience.blogspot.com
  20. Process Scale Up www.technologyinscience.blogspot.com
  21. • Bio-Resource www.technologyinscience.blogspot.com www.technologyinscience.blogspot.com

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