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Overexpression of cloned genes can yield large amounts of valuable proteins.

Overexpression of cloned genes can yield large amounts of valuable proteins. Genes can be overexpressed (1) in bacteria (e. g. E. coli ) (2) in eukaryotic cells. Many human proteins are used to treat disease. These are normally present in the body in trace amounts.

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Overexpression of cloned genes can yield large amounts of valuable proteins.

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  1. Overexpression of cloned genes can yield large amounts of valuable proteins. Genes can be overexpressed (1) in bacteria (e. g. E. coli ) (2) in eukaryotic cells

  2. Many human proteins are used to treat disease. These are normally present in the body in trace amounts. Protein Disease -antitrypsin Emphysema Epidermal growth factor Wound healing Insulin Diabetes Erythropoietin Anaemia Factor VII Haemophilia Factor IX Haemophilia Growth hormone Growth disorders Tissue plasminogen activator Heart attacks

  3. E. coliexpression plasmids Strong promoter (regulatable) Unique restriction sites e. g. Hin dIII CATATG Strong RBS Nde I site

  4. NNNN CAT ATG GGG ATG CCC TAC TGA AAA ACT TTT ACT TCGAA NNNN PCR is used to create an Nde I restriction site at the start codonof the target gene and another restriction site e. g. Hin dIII downstream of the stop codon.

  5. NNNCAT ATG TGA AGCTT NNN The amplified form of the gene (the PCR product) has the restriction sites at each end. Nde I Hin dIII

  6. NNNCAT ATG TGA AGCTT NNN CATATG Nde I Hin dIII Inserting the amplified gene into the expression vector places the start codon at the optimum distance from the RBS (2 - 11 bp upstream).

  7. The amplified gene must be re-sequenced to ensure that there are no base changes. Many heat-stable DNA polymerases have a high error rate. Taq polymerase has no proof-reading exonuclease activity and frequently mis-incorporates bases.

  8. The pT7-7 system pGP1-2 (KanR) Inducible T7 RNA polymerase Nde I HindIII T7 promoter (AmpR) pT7-7 expression plasmid

  9. T7 RNA pol - only recognises the T7 promoter (a 23 bp sequence) - initiates frequently - transcribes DNA at 200bases/sec (4 x faster than E. coli RNA pol)

  10. E. coli cells MW markers Control 205 + expression plasmid kDa 45 29 Band of overproduced protein. Analysis of expression level by protein gel electrophoresis.

  11. Band of overproduced protein.

  12. Target protein can be produced at a high level [ up to 30% of total cellular protein] Problems: Overproduced proteins may form inclusion bodies, aggregates of incorrectly folded inactive protein. Eukaryotic genes may have codon usage which is unfavourable for expression in E. coli. Specialised host strains have plasmids with extra copies of genes for rare tRNAs.

  13. Protein stability Recombinant proteins do not accumulate if they are rapidly degradedby proteases. The N-terminal AA affects stability. N-terminal AAHalf-life Met, Ser, Ala, Thr, Val, Gly 20 hours Ile, Glu > 30 min Tyr, Gln ~ 10 min Pro ~ 7 min Phe, Leu, Asp, Lys ~ 3 min Arg ~ 2 min Changing the first AA can improve stability.

  14. ATG CAC CAC CAC CAC CAT CAT ATG TAC GTG GTG GTG GTG GTA GTA TAC Some expression vectors encode hexahistidine tags. M H H H H H H M RBS Start codon Nde I Tagged proteins bind specifically to immobilised metal ion affinity columns. They can be purified in one step.

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