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