1. Extraction from natural system Required large quantities 80 μg of, the pure and biologically-active human hormone secretin, would require 3000 kg of bovine intestine. Difficult to meet the demand The quantity of purified insulin obtained from one pig satisfies the requirements of one diabetic person for three day Contamination
2. Commercial production of gene products requiresvery high expression levelsFactors that affect gene expression can be manipulated toincrease the amount of protein production Move gene to specialized expression vectors and hosts Location of gene (vector or hosts chromosome) Gene dosage Transcription • Promoter and terminator sequences • Regulatory genes and sequences
3. Translation • Ribosome binding site (Shine-Delgarno sequence) • Codon optimization to match hosts codon bias Final location of gene product • Cytoplasmic or extracellular (secreted out of cell) Protein stability • Degradation by host proteases (fusion protein) • Formation of insoluble aggregates
4. TranscriptionPromoters and enhancers are two major regulatory regionsthat controlled the transcription.Promoter contains specific DNA sequences that act as‘molecular switches’ to turn on transcription, in the upstreamdirection of the genes.Eukaryotic genes have enhancers in addition to promoters.Enhancers enhances transcription and are functional in anyorientation.
5. Types of promoter to regulate gene expressionConstitutive promoters Direct expression in virtually all tissues and are largely, if not entirely, independent of environmental factors.Tissue-specific promoters Direct expression of a gene in specific tissue or at certain stages of development.Chemical-inducible promoters Their performance can be controlled artificiallySynthetic promoters Made of the primary elements of a promoter region
6. The most commonly used promoters in E. coli
7. Translation The ribosome binding site (RBS)• Vary between E. coli strains• A consensus sequence (Shine-Delgarno sequence) is centered ~ 10 nucleotides upstream from the start codon.• Most sites contain the minimal sequence ***AGGA****• Base pairing between a mRNA’s Shine Delgarno sequence and the ribosome to select the proper initiation codon• If a eukaryotic gene to be expressed in E.coli, is cloned starting from the its first codon, a Shine-Delgarno sequence will have to be inserted upstream for the translation to occur
8. Codon Optimization
9. Final Yield of ProteinExpression of protein in secretion vectorProteins in periplasm easier to purifyProtein often more stable in periplasmSecretion to periplasm requires signal sequence
10. Protein stability: Fusion Proteins Recover from DNA culture medium and purify peptide protein of interestmRNA Maltose-binding protein
11. Fusion Proteins
12. Poor expression ?Presence of introns in eukaryotes : has been solved by using cDNAprepared from mRNADegradation of foreign proteins: prevented by using proteasedeficient strain or by fusion protein technique.Incorrect folding:leading to insoluble protein aggregates (inclusion bodies).Fusing to E. coli protein thioredoxin can increase solubility up to 40%of total cellular protein
13. Insoluble proteins are not a curse The blessings of inclusion bodies• Easy to segregate proteins in inclusion bodies by centrifugation.• Inclusion bodies protect protein from proteases.• Toxic proteins won’t kill the host cells before enough is expressed to be useful. On the other hand• Protein must be dissolved out of the inclusion bodies to be purified.• Inclusion body proteins are usually not folded completely; finishing the job can be a challenge.• Refolding is seldom complete and the portion of the protein which is active may be very low at best.
14. Problems with E. coli Endotoxins contamination Not able to do post-translational modifications likeGlycosylation Required for,Fatty acid acylation Proper folding of the secondary, tertiary andPhosphorylation and quaternary structures ofDisulfide-bond formation interested proteins These modifications can affect thebioactivity, function, structure, solubility, stability, half-life,protease resistance, and compartmentalization of thefunctional proteins.