1. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 The Cell as a Production Factory, Expression Systems Tobias Schoep

2. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 No chapter reference. Biology background, Chapter 15 Questions to schoep@engineering.ucsb.edu, Rm 3114 or contact Serra Elliot

3. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11

4. Cells as production factories Cellular method of protein production depends on protein properties Different systems have different advantages and disadvantages for protein production To understand the advantages and disadvantages of expression systems we must understand a bit more biology Biology of protein folding, transport and modification – a brief overview Prokaryotic protein expression systems Eukaryotic protein expression systems

5. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Protein Production Prokaryotes: Translation in the cytoplasm Eukaryotes: Translation in the cytoplasm and endoplasmic reticulum

6. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Protein Folding (Eukaryotes) Proteins are produced in cytosol and endoplasmic reticulum Endoplasmic reticulum targeting requires a signal sequence

7. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Protein Folding (Eukaryotes) Endoplasmic reticulm Function: Protein folding, glycosylation and export to golgi apparatus (eukaryotes) Correct folding eg. disulfide bond formation mediated by chaperones

8. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Protein Transport (Eukaryotes) Proteins move from ER to golgi apparatus in vesicles Golgi apparatus function: protein modification, packaging for distribution misfolded proteins

10. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Protein Transport (Eukaryotes) Lectures to come: Trafficking of proteins through cells Transport through membranes: facilitated and passive

11. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Protein Modification (Eukaryotes) Glycosylation Formation of protein / carbohydrate complex Important for protein structure, function and targeting Aberrant glycosylation can cause disease eg. Congenital Disorders of Glycosylation Many proteins glycosylated in ER

12. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Protein Modification (Eukaryotes) Glycosylation Additional modifications to glycoproteins in golgi apparatus

13. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Protein Folding (Prokaryotes) Proteins are produced in cytoplasm Cytoplasm is a mildly reducing environment Disulfide bond formation in periplasm Protein folding in cytoplasm can be mediated by chaperones

14. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Protein Transport (Prokaryotes) Protein transport is more complex in eukaryotes Transport is also important in prokaryotes eg. secreted proteins Targeting proteins to periplasm for disulfide bond formation Periplasmic targeting requires a signal sequence (signal peptide) MKK..HHHHHHHHHH.. Ala-X-Ala| -..........C’ Positively Charged N-term "AXA Box" Hydrophobic Region (10-20 aa)

15. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Protein Transport (Prokaryotes) There are at least 6 secretion systems Specific secretion systems are often bacterial species specific The type II secretion system (T2SS) is main secretion apparatus Sec /TAT /SRP complex

16. Protein Transport (Prokaryotes) In E.coli there are 3 types of T2SS from the cytoplasm to the periplasm Sec pathway: transport of unfolded proteins TAT pathway: transport of folded proteins SRP pathway: protein translated directly into periplasm Different signal sequences target proteins to secretion pathways ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Z= polar residue Φ = hydrophobic residues

17. Protein Modification (Prokaryotes) Most bacteria do not glycosylate If glycosylation is important in protein function, bacterially produced protein will not function ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11

18. SUMMARY As usual, eukaryotes are more complicated ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 cytoplasmic proteins ER proteins glycosylation disulfide formatin modification (eg.glycosylation) Packaging, distribution chaperones Recycle misfolded proteins

19. SUMMARY As usual, prokaryotes are complicated enough! ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Folded proteins TAT pathway Unfolded proteins Sec pathway SRP pathway Protein aggregation and degradation Disulfide formation Cytoplasmic proteins oxidised isomerase Chaperone

20. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Biology of protein production, folding and modification Prokaryotic protein expression systems Eukaryotic protein expression systems

21. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Prokaryotic protein expression systems Escherichia coli (E.coli) Used for production of first recombinant DNA biopharmaceutical (Insulin) by Eli Lilly Production of bovine growth hormone (bGH) on ton scale by Monsanto in 1994 ($11.60/g) (both Insulin and bGH require oxidative folding)

22. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Advantages of E.coli for protein production Rapid growth, on inexpensive carbon source Amenable to high density fermentation and scale-up Genetics very well characterized, chromosome sequenced Many tools for genetic manipulation/ cellular engineering Disadvantages of E.coli for protein production Cytoplasm is a mildly reducing environment Does not perform post-translational modifications eg. glycosylation Does not allow folding of complex proteins with multiple disufides Complex proteins often form inclusion bodies

23. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 What happens when proteins fold partially? Aggregate and may form inclusion bodies Degrade Eventually fold correctly

24. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Considerations when producing proteins in E.coli Gene and codon usage Transcriptional & Translation Regulation Protein Folding and Targeting Host Engineering: Chaperones & Proteases Culture Conditions

25. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Gene and codon usage Organisms show preference for codon (mRNA) that codes an amino acid Design genes to have optimal E. coli codon usage Codon usage reflects tRNA pool available for translation Produce codon optimized gene synthetically

26. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Transcriptional & Translation Regulation Low gene dosage/copy number Tightly-regulated promoters eg. arabinose operon Enhanced mRNA stability Optimized translational initiation eg. optmized Shine Dalgarno sequence

27. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Protein Folding and Targeting Cytoplasm is a mildly reducing environment Disulfide bond formation mainly in periplasm Protein folding in cytoplasm can be mediated by chaperones

28. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Protein Folding and Targeting Protein folding in cytoplasm can be mediated by chaperones Dna K- Dna J Release form Dna K Re-activate after stress Partially folded proteins Can form Inclusion bodies

29. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Protein Folding and Targeting Does the protein need to be secreted or expressed in the cytoplasm Cytoplamic expression- batch cultures Secreted proteins- continuous cultures Selection of appropriate secretion system considering disulfide formation requirements

30. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Protein Folding and Targeting Problems with secreted proteins: Incomplete processing of signal peptides Variable secretion efficiency Slow rate of accumulation (degradation?) Formation inclusion bodies Incorrect disulphide formation

31. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Host Engineering: Chaperones & Proteases Alter metabolism to favor protein production Metabolic engineering of pathways influencing protein production Eg. Slowing of glycolytic flux to reduce acetate formation Increase folding in periplasm Co expression of chaperones and foldases Eg. Seventeen Kd Protein (Skp) chaperone assists OMP folding, increased functional yield of scFv fragments in periplasm Eg. Protein Disulphide Isomerase

32. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Host Engineering: Chaperones & Proteases Reduce proteolytic degradation of proteins Knockout of protease genes Eg. Cytoplasmic Proteaes: Lon, Clp (A,X,Y,P, YQ) Eg. Periplasmic proteaes: DegP, Prc (Tsp) Eg. Membrane proteases: DegS, DegQ, Protease III, Increase folding in cytoplasm Engineering strains with altered redox environment in cytoplasm Eg. ORIGAMI (Novagen) allowed production of Human Tissue Plasminogen Activator with 17 dsbs.

33. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Culture Conditions Often protein specific Optimize substrate feed, temperature, induction conditions

34. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Biology of protein production, folding and modification Prokaryotic protein expression systems Eukaryotic protein expression systems

35. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11

36. Eukaryotic systems for protein production Yeast - Saccharomyces cerevisiae, Pichia pastoris Insect Cells (e.g., SF9) Mammalian Cells (CHO, NS0, MS2, Hybridoma) Plants Transgenic Animals

37. Considerations when producing proteins in eukaryotic systems Gene and codon usage – NO Transcriptional & Translation Regulation – YES – Tet ON, Tet OFF systems Protein Folding and Targeting – NO Host Engineering – YES Culture Conditions – YES Improved environmental control i.e., Temp, pH, fed-batch, media additives ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11

38. Yeast Unicellular organism As a eukaryote share the complex internal cell structure of plants and animals Can be grown in liquid culture like bacteria

39. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Advantages of Yeast (S.cerevisiae) for protein production Rapid growth, on inexpensive carbon source Amenable to high density fermentation and scale-up Genetics very well characterized, chromosome sequenced Many tools for genetic manipulation/ cellular engineering Can form correct disulfide bonds for eukaryotic proteins Can secrete proteins Disadvantages of Yeast (S. cerevisiae) Only simple glycosylation and hyperglycosylation (large mannose glycans are problematic for human therapeutics) Sometimes low expression levels (<mg/L)

40. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Host Engineering GlycoFi Introduce human glycosylation pathways in to Yeast (P. pastoris) Localized synthetic enzymes fusions in ER Produced complex human glycoproteins in yeast Technology purchased by Merk for $400M in 2006

41. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Mammalian cells Derived from a mammal Immortalized cells, such as cancer cells Generally adapted to suspension culture for protein production Common cell lines for protein production CHO – Chinese hamster ovary cells 293 - Human kidney 293 cells Hybridoma cells for antibody production (previous lecture 10/18/11)

42. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Advantages of mammalian cells for protein production Correct glycosylation of proteins (although can between cell types) Correct disulfide bridges formed Disadvantages of mammalian cells Slow growth rates Production of stable engineered cells takes up to 6 months Low yields (mg/L) Upscaling protein production can be problematic Some concerns with safety as many cell lines are cancer derived and viral vectors used for engineering

43. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Timeline for mammalian cells for protein production See lecture Manipulating genes, cellular engineering

44. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Host Engineering Metabolic engineering of pathways influencing protein production E.g. Reduction of lactate production. Lactic acid can inhibit cell growth and affect cellular metabolism at high concentrations. Metabolic engineering of pathways influencing cell survival E.g. Prevention of apoptosis (programmed cell death) by overexpression of Bcl-2. Inhibits formation of Mitochondrial Outer Membrane Permeabilization Pore.

45. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 CHO cells used for biologicals

46. ChE 170: Engineering Cell Biology –The cell as a production factory, expression systems – 11/10/11 Good luck in the quiz!