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Recombinant protein expression. Other alternatives

Recombinant protein expression. Other alternatives. Elvira Marín – Felipe Clemente WG1 - Protein expression UCM La Cristalera – Miraflores – 10/12/12. Unkown proteins cloned. Cloned in pANT7_cGST Dr. Manuel Fuentes. Chromosome 16. ̴ 260 Unkown proteins. WG1 25 new proteins.

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Recombinant protein expression. Other alternatives

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  1. Recombinant protein expression. Other alternatives Elvira Marín – Felipe Clemente WG1 - Protein expression UCM La Cristalera – Miraflores – 10/12/12

  2. Unkown proteins cloned Cloned in pANT7_cGST Dr. Manuel Fuentes Chromosome 16 ̴ 260 Unkown proteins WG1 25 new proteins Digest with restriction enzymes Sequencing Mass spectrometry (MRM) Expression (IVTT) and purification (GST)

  3. Unkown proteins UN-CLONED Chromosome 16 Unkown proteins Cloned in pANT7_cGST Design primers Gateway cloning system Master clone pDONR221 (recombinase)

  4. Unkown proteins uncloned pANT7_cGST (recombinase) Digestion and Sequencing Mass spectrometry (MRM) Expression (IVTT) and purification (GST)

  5. Protein expression

  6. Protein expression systems Prokaryotes Eukaryotes Bacteria Saccharomyces cerevisiae Pichia pastoris Escherichia coli CHO, HeLa, BHK Yeast Mammalian

  7. Protein expression systems LOW HIGH SPEED Mammalian Yeast Bacteria COST Bacteria Mammalian Yeast YIELD Mammalian Bacteria Yeast POST-TRANSLACTION MODIFICATION Yeast Bacteria Mammalian

  8. Protein expression systems E. coli Yeast Mammalian N.I. I M

  9. E. coli compartments Advantages Disadvantages Simple purification Proteolysisislessextensive Improvedfolding (S-S formation) Signaldoesnotalways facilitateexport Inclusionbodiesmay be formed Periplasm Inclusionbodies: easypurification protectionfromproteases inactive protein (non-toxic) Higherproteinyield (until 30% Biomass) Simplerplasmidconstruct Inclusionbodies: proteinfolding denaturation/refolding Cytoplasm Lessextensiveproteolysis Simple purification Improvedfolding Usually no secretion Celllysis Extracellular

  10. Fusion partners to the recombinant protein Maltose binding protein (MBP) Glutathione-S-transferase (GST) Small ubiquitin-modifier (SUMO) N-utilization substrate (NusA) Thioredoxin

  11. Pichia pastoris vs Saccharomyces cerevisiae Advantages P. pastoris and S. cerevisiae Short doubling time Readily manipulated genome Improved folding and post-translational modification Expression of similar genes and compatible vectors Better yield of recombinant protein (higher cell density) Methylotrophic yeast (methanol as its only carbon source) Strongly methanol induced promoters (alcohol oxidase genes: AOX1 and AOX2) Optimal growth pH 3.0-7.0 Extremely low levels of endogenous protein secretion Expression vectors integrated in the genome Disulfide bond formation and glycosylation modifications P. pastoris S. cerevisiae

  12. Expression on mammalian cells All posttranslational modifications Highest folding capacity (antibodies…) Secreted recombinant proteins for an easier purification rather than intracellular production Lowest yield Transfection more complex than plasmid transformation Stable or transient transfection (takes longer to obtain stable transformants)

  13. Protein expression systems summary Advantages Disadvantages Fastest expression method (days) Inexpensive bioproduction media and high density biomass Simple process scale-up Well characterized genetics Limited posttranslational modifications Unsoluble proteins and not correctly folded Bacteria Rapid expression method (weeks) Inexpensive bioproduction media and high density biomass Most posttranslational modifications High folding capacity N-linked glycan structures different from mammalian forms Yeast Transient-transfection Moderate rapid expression method (weeks) All posttranslational modifications and high folding capacity Low density biomass and expensive bioproduction media Difficult process scale-up Mammalian Stable-transfection All posttranslational modifications and high folding capacity Low density biomass and expensive bioproduction media Difficult process scale-up Longest expression method (months) Mammalian

  14. Elvira Marín – Felipe Clemente WG1 - Protein expression UCM Dra. Concha Gil Dr. Manuel Fuentes

  15. E. coli expression systems N.I. I M

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