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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. - PowerPoint PPT Presentation
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Application of biotechnology Expression in E. coli
Application of biotechnologyExpression in E. coliDr Muhammad ImranWe may not be aware but
Expression in E. coli needs signalsThree important signals needed for expression
PromoterRBSTranscription terminatorThree important signal elements
Promoter
Strong promotersWeak promotersConstitutive promoterRegulated promotersInduction and repression.Strong and week promoters
Induction and repression
MisfoldingForces that help protein fold.. H bonds, hydrophobic AA, ionic interactions, etcRate of transcription, translation and foldingFusion partner roleChaperon GroEl and GroES, DNAJKpHLigand for foldingDi-sulphide bonds1- Origami strain 2- Shuffle strain 3- Periplasmic localization signalHighlights
1- Constitutivelyexpresses 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 cytoplasmSignal/localization sequencesPeriplasm localization signalmRNA stabilityIt is normal cellular processmRNA formation and mRNA degradation determines the over all level at a given time.mRNA Length does matterSecondary structures
Rnase E mutatedToxic proteins C41Rare codonspRare 2 plasmid
Synthetic genesSecondary structure optimizedRare codon optimizedRnase cleavage site removedToxicityTight control on expressionExpression in stationary phasePre-proteinWeak promoterInclusion body and refolding Rifampicin blockingLeaky expressionpLysis S and L plasmidsArabad promoter strong repression
Solubility tagsPurification and detection tagsExpression in inclusion bodiesOptimization of expression strain media effect
Inducer concentration and temperature
OrganismsGram negative sources genes express better in E coli compared to gram positive organism sources