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

1. Recombinant Protein Expression
in E.coli
Bio-Resource
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2. Protein Expression in E.coli
• Procaryotic systems are well studied and widely used for
protein expression
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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.
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4. Protein Expression in E.coli
• Intra Cellular via Inclusion Bodies
• Expression Yield: 1 – 5 grams of Protein / Litre
• Examples: Insulin
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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)
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6. Vectors
• Vehicle / Carrier of Foreign DNA
Types of Vectors
• Plasmids,
• Viruses,
• Cosmids, and
• Artificial chromosomes
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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
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8. pUC19 Vector
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9. Most widely used vectors
• pET series from Novagen
• pALTER from Promega
• pCal-n from Stratagene
• pBAD/His from Invitrogen, etc
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10. Cloning & Screening
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11. Plasmid Extraction / Isolation
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Qiagen Plasmid Extraction Kit
Plasmid Extraction Procedure

12. Restriction Digestion & Ligation
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13. Restriction Digestion and Gel
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14. Transformation and Expression
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15. Transformation - Procedure
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16. Shake Flask Culture-
Protein Expression & Analysis
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17. Lac Operon & IPTG Induction
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18. Protein Analysis – SDS PAGE
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19. Protein Characterization
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20. Process Scale Up
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21. • Bio-Resource
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