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Application of biotechnology Expression in E. coli Dr Muhammad Imran.

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Presentation on theme: "Application of biotechnology Expression in E. coli Dr Muhammad Imran."— Presentation transcript:

1 Application of biotechnology Expression in E. coli Dr Muhammad Imran

2 We may not be aware but……

3

4 Expression in E. coli needs signals Three important signals needed for expression Promoter RBS Transcription terminator

5 Three important signal elements

6 Promoter

7 Strong promoters Weak promoters Constitutive promoter Regulated promoters Induction and repression.

8 Strong and week promoters

9 Induction and repression

10 Misfolding Forces that help protein fold.. H bonds, hydrophobic AA, ionic interactions, etc Rate of transcription, translation and folding Fusion partner role Chaperon GroEl and GroES, DNAJK pH Ligand for folding

11 Di-sulphide bonds 1- Origami strain 2- Shuffle strain 3- Periplasmic localization signal Highlights 1- Constitutively expresses a chromosomal copy of the disufide bond isomerase DsbC 2- DsbC promotes the correction of mis-oxidized proteins into their correct form (1,3) 3- The cytoplasmic DsbC is also a chaperone that can assist in the folding of proteins that do not require disulfide bonds (4) 4- DsbA in periplasm express in cytoplasm 5- thioredoxins and glutaredoxins reductaces maintain a reducing environment in cytoplasm

12 Signal/localization sequences Periplasm localization signal

13 mRNA stability It is normal cellular process mRNA formation and mRNA degradation determines the over all level at a given time. mRNA Length does matter Secondary structures Rnase E mutated Toxic proteins C41

14 Rare codons pRare 2 plasmid Synthetic genes Secondary structure optimized Rare codon optimized Rnase cleavage site removed

15 Toxicity Tight control on expression Expression in stationary phase Pre-protein Weak promoter Inclusion body and refolding Rifampicin blocking

16 Leaky expression pLysis S and L plasmids Arabad promoter strong repression

17 Solubility tags

18 Purification and detection tags

19 Expression in inclusion bodies

20 Optimization of expression strain media effect

21 Inducer concentration and temperature

22 Organisms Gram negative sources genes express better in E coli compared to gram positive organism sources

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