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

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Application of biotechnology Expression in E. coli Dr Muhammad Imran

We may not be aware but……

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

Three important signal elements

Promoter

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

Strong and week promoters

Induction and repression

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

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

Signal/localization sequences Periplasm localization signal

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

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

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

Leaky expression pLysis S and L plasmids Arabad promoter strong repression

Solubility tags

Purification and detection tags

Expression in inclusion bodies

Optimization of expression strain media effect

Inducer concentration and temperature

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