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RECOMBINANT PROTEINS

Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s P rogrammes at the University of Pécs and at the University of Debrecen Identification number : TÁMOP-4.1.2-08/1/A-2009-0011.

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RECOMBINANT PROTEINS

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  1. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

  2. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Beáta Scholtz Molecular Therapies- Lecture 3 RECOMBINANT PROTEINS

  3. RECOMBINANT PROTEINS The aim of this lecture is to describe the in vitro and in vivo systems utilized for expression of recombinant proteins, and discuss the advantages and disadvantages of these systems. We will also discuss the basics of affinity-tag based protein purification. 1.1 OVERVIEW: PROTEIN PHARMACEUTICALS 1.2 CELL-FREE SYSTEMS: IN VITRO TRANSCRIPTION AND TRANSLATION 1.3 EXPRESSION OF RECOMBINANT PROTEINS IN CELL CULTURE 1.4 NON-PROKARYOTIC EXPRESSION SYSTEMS 1.4.1 Pichia pastoris 1.4.2 Protein expression in insect cells 1.4.3 Mammalian expression systems 1.5 PURIFICATION OF RECOMBINANT PROTEINS

  4. TÁMOP-4.1.2-08/1/A-2009-011 Pure protein preparations Uses: medicine and research Sources: • natural protein mixtures - human/animal/fungi/plant • artificial preparations - synthetic peptides, recombinant proteins Protein pharmaceuticalNatural Source Insulin Pigs or cattle (pancreas) Factor VIII Human blood (donated) Human growth hormone Human brains Calcitonin Salmon Anti-venom Horse or goat blood

  5. TÁMOP-4.1.2-08/1/A-2009-011 Equipment used for blood fractionation

  6. TÁMOP-4.1.2-08/1/A-2009-011 Antivenom - specific antiserum from goat or horse Box jellyfish, Australia B. Rogge P-A. Olsson R. Morante Black scorpion, Arabia Lonomia caterpillar, Brasil

  7. TÁMOP-4.1.2-08/1/A-2009-011 Protein pharmaceuticals • Natural sources are often rare and expensive • Difficult to keep up with demand • Hard to isolate product • May lead to immune reactions (diff. species) • Viral & pathogen contamination • Most protein pharmaceuticals today are produced recombinantly • Cheaper, safer, abundant supply

  8. TÁMOP-4.1.2-08/1/A-2009-011 Peptide drugs • Many hormones are actually small peptides (2-40 amino acids) • Calcitonin (32 residues) • Thyroid hormone to enhance bone mass • Oxytocin (9 residues) • Pituitary hormone to stimulate labor • Vasopressin (9 residues) • Pituitary hormone for antidiuretic/vasoconstriction

  9. TÁMOP-4.1.2-08/1/A-2009-011 Peptide drugs • Small enough to synthesize using solid phase chemistry (SPPS) • Method developed by Bruce Merrifield in 1960’s (won Nobel prize) • Very efficient synthesis (>99%/couple) • Still: 50 residue peptide, 99% coupling • Yield = 0.9950 = 60.5% • Technique limited to small peptides

  10. TÁMOP-4.1.2-08/1/A-2009-011 Recombinant proteins • Developed in 1970’s &1980’s • Paul Berg (1973) restriction enzymes • Herbert Boyer (1978) cloning human insulin into E. coli – Genentech • Four general approaches • Expression in cell-free systems • Expression in isolated cells • Expression in transgenic plants/animals • Gene therapy in humans

  11. TÁMOP-4.1.2-08/1/A-2009-011 Cell-free systems:In vitro transcription and translation • Rapid identification of gene products • Functional analyses • Analyze protein-protein interactions • Study protein folding • Incorporate modified amino acids for functional studies • Engineer truncated gene products

  12. TÁMOP-4.1.2-08/1/A-2009-011 Cell-free systems:In vitro transcription and translation • Advantages over in vivo gene expression: • When the protein is: • toxic to the host cell • insoluble or forms inclusion bodies • degraded rapidly by intracellular proteases • Speed and directness of all procedures • Absence of constraints from a living cell • Pure product • Disadvantages over in vivo gene expression: • Lack of cellular membranes • Lack of post translational modifications

  13. TÁMOP-4.1.2-08/1/A-2009-011 Components for in vitro transcription In vivo In vitro • Linearized DNA template • Phage RNA polymerase • 4dNTP • Buffer 1998 by Alberts, Bray, Johnson, Lewis, Raff, Roberts, Walter. Published by Garland Publishing.

  14. TÁMOP-4.1.2-08/1/A-2009-011 Phage RNA polymerases

  15. TÁMOP-4.1.2-08/1/A-2009-011 Characteristics of RNA polymerases • RNA polymerases proceed at a much slower rate than DNA polymerases. • RNA pol (50-100 bases/sec) • DNA pol (1000 bases/sec) • The fidelity of RNA synthesis is much lower than that of DNA. • RNA polymerases do not contain proofreading mechanisms.

  16. TÁMOP-4.1.2-08/1/A-2009-011 DNA template • Plasmids • Many commonly used cloning vectors contain phage polymerase promoters outside of the multiple cloning site. • PCR Products • Primer must • contain promoter • Oligonucleotides

  17. TÁMOP-4.1.2-08/1/A-2009-011 Linearization of template • Plasmids: no RNA polymerase termination signal; templates are linearized • PCR template: termination signal in the amplified region OR in the primer

  18. TÁMOP-4.1.2-08/1/A-2009-011 Translation in eukaryotic cells 1998 by Alberts et al. Published by Garland Publishing.

  19. TÁMOP-4.1.2-08/1/A-2009-011 Components for in vitro translation • tRNA & aminoacyl-tRNA synthetases • Ribosomes • Amino acids • ATP, GTP • Initiation, elongation, and termination factors • Buffer • RNA template Much more complex than transcription Cannot be mixed from a few isolated components Always provided as crude extract of cells

  20. TÁMOP-4.1.2-08/1/A-2009-011 Common in vitro translation systems • Rabbit reticulocyte lysate • Wheat germ extract • E. coli extract

  21. TÁMOP-4.1.2-08/1/A-2009-011 Rabbit reticulocyte lysates • Reticulocytes: • immature red blood cells • no nuclei (DNA) • complete translation machinery, for extensive globin synthesis • Endogenous globin mRNA can be eliminated by incubation with a Ca2+dependent micrococcal nuclease. The nuclease is later inactivated by EGTA. • Low background • Efficient utilization of exogenous RNAs, even at low concentrations • Low nuclease activity • Capable of synthesizing large amounts of full-length products • Capable of translating either capped or uncapped RNAs

  22. TÁMOP-4.1.2-08/1/A-2009-011 Wheat germ lysates • Low background incorporation due to low levels of endogenous mRNA • Recommended for translation of RNA containing small fragments of double-stranded RNA or oxidized thiols, which are inhibitory to the rabbit reticulocyte lysate • Generally more cap dependent than reticulocyte systems • Often preferable when synthesizing relatively small proteins (12-15kDa) that comigrate with globin, which is abundant in reticulocyte extracts

  23. TÁMOP-4.1.2-08/1/A-2009-011 E. coli lysates • Simple translational apparatus and less complicated initiation control mechanisms • BUT: bacterial extracts contain nucleases that rapidly degrade most exogenous RNAs • Extract must be incubated during preparation so that excess endogenous mRNA is translated and subsequently degraded • The exogenous product is easily identifiable

  24. TÁMOP-4.1.2-08/1/A-2009-011 Translation systems • Two approaches to cell free protein synthesis: • Standard translation systems (reticulocyte and wheat extracts) use RNA as a template • Linked or coupled transcription+translation systems start with DNA templates • Important elements for translation: • = Eukaryotic translation signal: 5’-GCCACCAUGG-3’ “Kozak” sequence, if eukaryotic cell free translation system is used • = Prokaryotictranslation signals: 5’-UAAGGAGGUGA-3’ Shine- Delgarno (SD) , if prokaryotic cell free translation system is used • Linked system: tube 1.=transcription, tube 2.= translation. • = Each can be optimized separately. • Coupled system: both reactions in the same tube

  25. TÁMOP-4.1.2-08/1/A-2009-011 Main steps of recombinant protein production In vivo Cell free Identification/Isolation of gene of interest Cloning of gene into plasmid Plasmid: expression vector Transformation into host cells Growth of cells through fermentation Plasmid: source of DNA template for transcription In vitro transcription In vitro translation Isolation & purification of protein Formulation of protein product Research

  26. TÁMOP-4.1.2-08/1/A-2009-011 Recombinant protein expression in cells or organisms • Escherichia coli/ Other bacteria • Pichia pastoris/ Other yeast • Insect cell culture (Baculovirus) • Mammalian cell culture • Plants • Sheep/cows/humans • (transgenics and gene therapy)

  27. TÁMOP-4.1.2-08/1/A-2009-011 Expression system selection • Choice depends on size and character of protein • Large proteins (>100 kD)? Choose eukaryote • Small proteins (<30 kD)? Choose prokaryote • High yields, low cost? Choose E. coli • Post-translational modifications essential? Choose yeast, baculovirus or other eukaryote • Glycosylation essential? Choose baculovirus or mammalian cell culture

  28. TÁMOP-4.1.2-08/1/A-2009-011 Characteristics of (plasmid) vectors • 1. Must be compatible with host cell system (prokaryotic vectors for prokaryotic cells, eukaryotic vectors for eukaryotic cells) • 2. Features : • strong promoter/inducible promoter • transcription START sequences • ribosome binding sites • termination sequences, polyA signal sequence • affinity tag or solubilization sequences • multi-enzyme restriction site • origin of replication (ORI) • bacterial selectable marker (Amp or Tet) • eukaryotic selectable marker • recombination sequences protein expression cloning, plasmid propagation

  29. TÁMOP-4.1.2-08/1/A-2009-011 Promoter selection • Constitutive - everywhere, all the time • Tissue- or developmental stage-specific - selected cell types, • specific timing • Inducible - specific timing, • can avoid toxicity to host • Synthetic

  30. TÁMOP-4.1.2-08/1/A-2009-011

  31. TÁMOP-4.1.2-08/1/A-2009-011 Inducible promoters: Tet-off system

  32. TÁMOP-4.1.2-08/1/A-2009-011 Inducible promoters: Tet-on system (faster response)

  33. TÁMOP-4.1.2-08/1/A-2009-011 Synthetic promoters, inducible systems Steroid hormone induction: adenovirus promoter glucocorticoid response element inducer: dexamethasone Tetracycline operon: CMV promoter Tet operator sequence, Tet repressor protein inducer/repressor: tetracycline Ecdyson-inducible system: requires two vectors SV40 promoter human RXR receptor and Drosophila ecdyson receptor (VgEcR) = transcription factor heterodimer Activator of transcription factor: pronasteroneA Nice dose response

  34. Grows quickly (8 hrs to produce protein) High yields (50-500 mg/L) Low cost of media (simple media constituents) Low fermentor costs Difficulty expressing large proteins (>50 kD) No glycosylation or signal peptide removal Eukaryotic proteins are sometimes toxic Can’t handle S-S rich proteins TÁMOP-4.1.2-08/1/A-2009-011 Bacterial expression systems AdvantagesDisadvantages

  35. TÁMOP-4.1.2-08/1/A-2009-011 Promoter selection for prokaryotes

  36. TÁMOP-4.1.2-08/1/A-2009-011 Cloning & transforming in yeast cells Pichia pastoris Saccharomyces cerevisiae

  37. TÁMOP-4.1.2-08/1/A-2009-011 Pichia pastoris • Yeast are single celled eukaryotes • Behave like bacteria, but have key advantages of eukaryotes • P. pastoris is a methylotrophic yeast that can use methanol as its sole carbon source (using alcohol oxidase) • Has a very strong promoter for the alcohol oxidase (AOX) gene (~30% of protein produced when induced)

  38. Grow quickly (8 hrs to produce protein) Very high yields (50-5000 mg/L) Low cost of media (simple media constituents) Low fermentor costs Can express large proteins (>50 kD) Glycosylation & signal peptide removal Has chaperonins to help fold “tough” prtns Can handle S-S rich proteins TÁMOP-4.1.2-08/1/A-2009-011 Pichia expression system AdvantagesMore advantages

  39. TÁMOP-4.1.2-08/1/A-2009-011 Pichia pastoris cloning • Uses a special plasmid that works both in E. coli and yeast • Once gene of interest is inserted into this plasmid, it must be linearized • Transfect yeast cells with linear plasmid • Double cross-over recombination event occurs to cause the gene of interest to insert directly into P. pastoris chromosome where the old AOX gene used to be • Now gene of interest is under control of the powerful AOX promoter • Stable transfectant

  40. TÁMOP-4.1.2-08/1/A-2009-011 Cloning a gene into Pichia vector

  41. TÁMOP-4.1.2-08/1/A-2009-011 Baculovirus/insect cell expression systems Spodoptera frugiperda Spodoptera f. larva Bastiaan (Bart) Drees Sf9 cells and baculovirus

  42. TÁMOP-4.1.2-08/1/A-2009-011 Baculovirus life cycle 2. 4a. 3a. 3b. 4b. 1.

  43. TÁMOP-4.1.2-08/1/A-2009-011 Baculovirus life phases in culture Early phase: cell entry, shutting down host gene expression viral protein synthesis Late phase: viral DNS replication, virus assembly, release of viral particles from cell (peak:18-36 hrs post-infection) Also used to prepare viral stock Very late phase: polyhedrin and p10 genes are expressed, viruses embedded in polyhedrin form occlusion bodies. Cell lysis. (24-96 hrs post-infection) Used for protein production

  44. TÁMOP-4.1.2-08/1/A-2009-011 Baculovirus mediated protein expression in insect cells • Autographica californica multiple nuclear polyhedrosis virus (Baculovirus) • Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae) • Uses super-strong promoter from the polyhedrin coat protein to enhance expression of proteins while virus resides inside the insect cell - protein is not required for infection or viral life cycle • Secreted proteins better expressed by stably transfected insect cell lines, from the ie-1 promoter • (infection interferes with secretory pathways)

  45. TÁMOP-4.1.2-08/1/A-2009-011 Baculovirus expression system workflow Cloning gene of interest into baculovirus genome Use recombinant baculoviral DNA to transfect insect cells Collect viral particles from insect cell culture supernatant Test viral stock titer, freeze stocks Infect new insect cell culture Harvest cells (with occlusion bodies) Note: not a stable cell line!

  46. Transfer vector 5’ 3’ x x TÁMOP-4.1.2-08/1/A-2009-011 Cloning a gene into baculovirus (AcMNPV) vector Site-specific transposition Cloned gene Cloned gene 5’ 3’ Recombinant AcMNPV bacmid modified AcMNPV DNA, “Bacmid” maintained in E. coli

  47. Tn7R polyhedrin promoter Gent+ Tn7L Gene of Interest Transfer vector with insert Gene of Interest PpH Tn7 L Tn7 R Bacmid with insert TÁMOP-4.1.2-08/1/A-2009-011

  48. 128bp 145bp Mini att Tn7 M 13 reverse M 13 forward TÁMOP-4.1.2-08/1/A-2009-011 Tn7R GOI Tn7L Transposition into bacmid Bacmid DNA

  49. Grow very slowly (10-12 days for set-up) Cell culture is only sustainable for 4-5 days Set-up is time consuming, not as simple as yeast Can express large proteins (>50 kD) (Mostly) Correct glycosylation & signal peptide removal Has chaperonins to help protein folding Very high yields, cheap TÁMOP-4.1.2-08/1/A-2009-011 Baculovirus expression system DisadvantagesAdvantages

  50. TÁMOP-4.1.2-08/1/A-2009-011 Baculovirus successes • Alpha and beta interferon • Adenosine deaminase • Erythropoietin • Interleukin 2 • Poliovirus proteins • Tissue plasminogen activator (TPA)

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