Recombinant protein expression.pdf

Classic techniques of molecular biology and recombinant DNA technology Classic paradigm of molecular biology Amino acid sequence of protein determines its secondary, tertiary and quaternary structure (Anfinsen, 1972) Relationship of "molecular-scale" biological sciences There is not a hard-lin...

Classic techniques of molecular biology and recombinant DNA technology Classic paradigm of molecular biology Amino acid sequence of protein determines its secondary, tertiary and quaternary structure (Anfinsen, 1972) Relationship of "molecular-scale" biological sciences There is not a hard-line between these disciplines as there once was. Tools of classic molecular biology • Restriction enzymes • Electrophoresis • Polymerase chain reaction (PCR) • DNA sequencing • Blotting Paul Berg Herbert Boyer Stanley Cohen Berg, Boyer and Cohen developed techniques for locating, isolating, preparing, and studying small segments of DNA derived from much larger chromosomes Kary Mullis Types of restriction enzymes Type I - need ATP ➢ cleave DNA at random sites that can be >1kbp from recognition sequence Type III - need ATP ➢ cleave DNA ~25 bp from recognition sequence Type II - don’t require ATP ➢ Cleavage site is within recognition sequence ➢ Can produce sticky end or blunt end cuts Most restriction enzymes recognize short palindromes and cut unmethylated DNA Frequency of 6 cutters: 46 = once/4096 bp Frequency of 4 cutters: 44 = once/256 bp Agarose gel electrophoresis Pattern of DNA fragments produced by restriction enzyme can serve as a fingerprint of a DNA molecule e.g., fragments produced by cleaving SV40 DNA with each of three restriction enzymes Restriction Fragment Length Polymorphism (RFLP) resulting from βglobin gene mutation • In normal cells, sequence corresponding to 5th - 7th aa’s of the βglobin peptide is CCTGAGGAG; recognized by the restriction enzyme MstII • In the sickle cell, one base is mutated from A to T, making the site unrecognizable by MstII Application of PCR based - RFLP for species identification of ocular isolates of methicillin resistant staphylococci (MRS) Indian J Med Res 130, July 2009, pp 78-84 Fig. 3. Agarose gel electrophoresis of fragments produced by AluI digestion of 933-bp PCR amplification products from Staphylococcus species. Lane 1: negative control; Lane 2: Undigested product (933 bp); Lane 3: S.saprophyticus lab isolate; Lane 4: S. haemolyticus; Lane 5: S. xylosus; Lane 6: S. hominis; Lane 7: S. cohnii subsp. urealyticum; Lane 8: S. equorum; Lane 9: S. epidermidis; Lane 10: S. aureus; Lane 11: S. aureus (ATCC 6538); Lane 12: S. epidermidis (ATCC 12228); Lane 13: 100 bp ladder. Application of PCR-RFLP analysis on species identification of canned tuna Recombinant DNA/Molecular clones • DNA sequences that combine genetic material from multiple sources • Applications include: ❑Producing a specific gene/DNA fragment of interest for further study ❑Expression of a protein product Molecular cloning Requires two elements: 1.DNA fragment to be propagated ❑ may encode a gene of interest 2.Vector – DNA molecule in which target gene will be inserted ❑ needs to be able to self replicate Polylinkers: Inserted DNA fragments with multiple recognition sequences Cut target DNA segment using a specific restriction enzyme Insert into a vector e.g., the plasmid pUC18 DNA cloning vectors 1. plasmids ❑can carry up to 20 kb of foreign DNA 2. cosmids – can carry up to 45 kb of foreign DNA ❑can be packaged in lambda phage particles for infection into E. coli 3. bacteria artificial chromosomes (BACs) ❑carry 100 – 300 kb DNA inserts 4. yeast artificial chromosomes (YACs) ❑can carry up to 1 MB of foreign DNA Plasmids • Self-replicating, extrachromosomal circular DNA molecules • Usually nonessential for cell survival • Used as a cloning vector for small pieces of DNA (typically 50 to 5000 base pairs) Selection of the plasmid vector: purpose of use Purpose Special vector feature(s) Example Recombinant protein expression in bacteria Regulated bacterial promoter Tag for protein purification pGEX4T Recombinant protein expression in eukaryotic cells Eukaryotic promoter pcDNA3.1 Tag for protein purification or detection Eukaryotic selection marker Reporter gene pGL3basic Analysis of eukaroytic promoter General cloning - pBluescript KS Bacterial artificial chromosomes (BACs) as cloning vectors Recall lac operon If gene is inserted, no β-gal produced; colonies white instead of blue Cut EcoRI / Pvu II / Not I DNA Marker Cut EcoRI / Pvu II Cut EcoRI / Pvu II Cut EcoRI / Pvu II Insert DNA Marker Distance DNA Marker Log10 bp Analysis of recombinant clones: restriction enzyme digestion 1.2% agarose gel cast In 1X TAE buffer DNA fragments stained with ethidium bromide and visualized by UV illumination. EcoR I Pvu II Not I Vector Insert Essential vector elements ❑ Origin of replication ❑ Antibiotic resistance gene (e.g., Amp, Kan, Tet, Chl) ❑ Multiple cloning site ❑ Promoter Map of pOTB7 vector showing Chloramphenicol resistance gene (CMR), replication origin (ORI) and multiple cloning site (MCS) Uses of cloned DNA Used in subsequent steps in the experimental design ❑PCR ❑Sequencing ❑Subcloning ❑Mutagenesis ❑Transfection into eukaryotic cell lines ❑Fragment isolation for transgenic mice production (microinjection) Expression cloning Introducing DNA into bacterial cells = transformation ❑ can be done using several methods, e.g., electroporation microinjection passive uptake conjugation Introducing DNA into eukaryotic cells, such as animal cells = transfection ❑Several different transfection techniques are available e.g., calcium phosphate transfection, liposome transfection ❑DNA can also be introduced into cells using viruses or pathogenic bacteria as carriers Recombinant protein expression Modern protein production usually use recombinant methods Natural sources are often rare and expensive ❑Difficult to keep up with demand ❑Hard to isolate product ❑Lead to immune reactions (diff. species) ❑Viral/pathogen contamination; e.g., human growth hormone Most protein pharmaceuticals today are produced using recombinant methods ❑Cheaper, safer, abundant supply Steps in recombinant protein purification ❑ Design expression plasmid, transform, select ❑ Grow culture of positive clone, induce expression ❑ Lyse cells ❑ Centrifuge to isolate protein-containing fraction ❑ Column Chromatography—collect fractions ❑ Assess purity on SDSPAGE Vector selection Must be compatible with host cell system ❑prokaryotic vectors for prokaryotic cells ❑eukaryotic vectors for eukaryotic cells Needs a good combination of ❑Inducible strong promoters ❑ribosome binding sites ❑termination sequences ❑affinity tag or solubilization sequences ❑Polylinker site Expression vectors for E. coli: examples pBAD – Dose-responsive induction by addition of arabinose – highly inducible: possible to achieve >1000-fold induction upon removal of glucose, addition of Larabinose ara C = positive/negative regulator • When arabinose is present, it binds to ara C causing it to change shape • New shape promotes attachment of RNA polymerase, txn initiation Transcriptional regulation using lac operon system Replace 3 genes with gene of interest! IPTG used in the lab for inducing gene expression in engineered bacterial strains: • mimics effect of lactose • not metabolized • longer lasting • provides strong induction of lac operon Lactose IPTG pET vector • Can produce recombinant protein up to 50% of total cell protein • contains T7 promoter adjacent to lac operator Host bacteria typically E.coli BL(DE3) ❑ Host T7 RNA polymerase under control of lac promoter/operator ❑ IPTG induction causes host to produce T7 RNA polymerase ❑ Phage T7 RNA polymerase specifically recognizes plasmid T7 promoter pGEX plasmid vector: • Gene encoding affinity tagglutathione S tranferase (GST) • Spacer between genes ❑encodes protease cleavage site (thrombin) • Ptac promoter ❑inducible with IPTG • Ribosome binding site Engineering proteins for ease of purification and detection ❑ Once you have a gene cloned and can over-express the protein, you can alter protein to improve the ease of purification or detection ❑ Can fuse a tag to the N-or C- terminus of your protein ❑ Can decide to remove the tag or not Basic strategies ❑ Add signal sequence that causes secretion into culture medium ❑ Add protein that helps the protein refold and stay soluble ❑ Add sequence that aids in precipitation ❑ Add an affinity handle (by far the most used is the His-tag) ❑ Add sequence that aids in detection Protein purification by column chromatography Ligand and metal binding • Affinity for cofactors, substrates, effector molecules, metals, DNA • When ligand is immobilized on a bead, you have an affinity bead Chromatographic modes of protein purification pGEX plasmid vector: • Gene encoding glutathione-S- transferase (GST) affinity tag ❑High affinity for glutathione • Ptac promoter ❑inducible with IPTG • Ribosome binding site • Spacer between genes ❑encodes thrombin cleavage site ❑Thrombin cleaves sequence between target protein and its tag Affinity chromatography: separation by biological binding interactions Example: GST - Glutathione • GST-tagged proteins bind to gluthatione on beads • • Non-specifically or weakly bound proteins washed off GST-tagged proteins eluted with glutathione (competitor) or thrombin (protease) Ligation inserts gene in-frame with GST tag In frame in pGEX-2T BamHI CTG GTT CCG CGT GGA TCC CCG GGA ATT CAT CGT GAC TGA CTG ACG L V P R G S P G I H R D * Insert into BamHI site BamHI insert BamHI CTG GTT CCG CGT GGA TCC CTG GGT GAG CGT GAA GCG GGA TCC CCG GGA ATT CAT CGT GAC TGA L V P R G S L G E R E A G S P G I H R D * Out of frame in pGEX-3X BamHI ATC GAA GGT CGT GGG ATC CCC GGG AAT TCA TCG TGA CTG ACT GAC I E G R G I P G N S S * Insert into BamHI site BamHI insert BamHI ATC GAA GGT CGT GGG ATC CCT GGG TGA GCG TGA AGC GGG ATC CCC GGG AAT TCA TCG TGA I E G R G I P G * A * S G I P G N S S * * indicates stop codon Affinity chromatography: tags for recombinant proteins GST = small enzyme that binds glutathione; fused to C-terminus of protein of interest GST-tagged protein binds to immobilized glutathione in affinity column http://www.cellmigration.org/resource/discovery/d iscovery_proteomics_approaches.html Commercially available protein purification kits GST•Bind™ Purification Kits His•Bind® Purification Kits Magnetight™ Oligo d(T) Beads MagPrep® Streptavidin Beads Protein A and Protein G Plus Agaroses S•Tag™ Purification Kits Streptavidin Agarose T7•Tag™ Affinity Purification Kit ProteoSpin™ CBED (Concentration, Buffer Exchange and Desalting) Maxi Kit — Effectively desalts and concentrates up to 8 mg of protein with an efficient, easy-to-use protocol.(Norgen Biotek Corporation) ProteoSpin™ Detergent Clean-up Micro Kit — Provides a fast and effective procedure to remove detergents including SDS, Triton® X-100, CHAPS, NP-40 and Tween 20. (http://www.emdbiosciences.com) How do we recognize target protein? Viable assay needed! ➢ Should target a unique identifying property of the protein • For enzymes, usually based on reaction it catalyzes in the cell ➢ Measure unit activity, where Unit = quantitative measure of activity usually associated with a turnover rate (for enzymes) or amount needed for stoichiometric binding (for receptors, ligands, DNA binding proteins) e.g., 1 Unit = 0.1 optical density increase in a 1 cm cuvette at 650 nm at 37° and pH 6.2 ➢ can be used to estimate amt of enzyme present in sample e.g., Lactate dehydrogenase assay • NADH absorbs light at 340 nm • assay for lactate dehydrogenase activity → increase in A340 observed in 1 minute • In general, no. of units/mg protein/ml increases increases with purity • Assays need to be relatively rapid, reproducible to be useful Problems with immunoaffinity chromatography Theoretically, can have a monoclonal antibody produced ➢ Stationary phase that will bind protein target Problems: 1. 2. 3. MAbs expensive, difficult to purify Other proteins may inactivate or bind non-specifically to them Some of the Mabs may leach off the column during the purification; must be removed Affinity columns usually used as an expensive last resort! – done late in the process when ❑volume has been reduced ❑majority of contaminants have already been removed Case study: recombinant DHFR production Dihydrofolate Reductase (DHFR): ~21 kDa enzyme ➢ catalyzes NADPH-dependent reduction of dihydrofolate to tetrahydrofolate ➢ DHFR inhibition, reduction disrupts nucleic acid, aa synthesis affecting Availability of purified DHFR facilitates ❑Cell growth identification of antifolate inhibitors with greater potency, higher selectivity ❑Proliferation GST-DHFR-His Construct GST – DHFR - His Glutathione-S-transferase ❑ Added to increase solubility ❑ Can be used as a secondary purification methodology Histidine tag ❑ 6 Histidine tag that binds to certain metals such as nickel Human dihydrofolate reductase ❑ Gene product of interest ❑ Target for chemotherapy reagents Protein expression and purification series workflow Streak Cells Overnight culture Subculture, monitor, and induce Harvest and lyse cells Purify Centrifugation or Instrumentation Analyze 45 Analysis of protein extract Protein quantitation (using A280) Beer’s Law A=ecl DHFR enzyme assay 46 Causes for getting inactive proteins • Requires molecular chaperone for proper folding • Requires post-translational modifications • Cofactors/protein partners needed for proper folding ➢ up to 1/3 of eukaryotic proteins may be “natively” unfolded until binding their protein partner • Need to obtain these proteins as correctly folded molecules! A good purification scheme takes into account both purification levels and yield. • A high degree of purification but poor yield leave little protein for experiments • A high yield with low purification leaves many contaminants in the fraction – complicates the interpretation of experiments. Bacterial systems Advantages • Grow quickly (8-12 hrs to produce protein) • High yields (50-500 mg/L) • Low cost of media (simple media constituents) • Low fermentor costs Disadvantages • Difficulty expressing large proteins (>50 kD) • No glycosylation or signal peptide removal • Eukaryotic proteins are sometimes toxic • Can’t handle S-S rich proteins Alternatives to bacterial protein production • Use different expression system • Use different host for protein expression ❑Yeast (Pichia pastoris) ❑Virus (Baculovirus) ❑Mammalian cell culture ❑Plants ❑Sheep/Cows Recombinant protein production Cloning and transforming in yeast cells Yeast = single-celled eukaryotes, but bacteria – like in ability to reproduce ❑many cDNA genes available ❑Glycosylation possible ❑Similar regulation as in bacteria; e.g., yeast GAL1 and GAL10 genes expressed when yeast cells are grown in media with galactose, but shut down in media with glucose Yeast strains used: ❑S. cerevisiae ❑Pischia pastoris - methylotrophic yeast; can use methanol as sole carbon source using alcohol oxidase (AOX) ➢very strong promoter for AOX gene ➢~30% of protein produced when induced Pichia pastoris cloning • Clone gene of interest in plasmid that works in E. coli, yeast • Linearized plasmid includes viral recombination region • Double cross-over recombination event occurs ➢ GOI inserted into P. pastoris chromosome Expression induced by MeOH Yeast systems Advantages • • • • • • • • Grow quickly (12-24 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 and signal peptide removal Has chaperonins to help fold “tough” proteins Can handle S-S rich proteins Baculovirus protein expression Baculoviruses = insect viruses with ds DNA genomes • act as parasites when they infect insect larval hosts; larvae become factories for virus production • p10 and polyhedrin, proteins produced in infection process, can be replaced by GOI • Recombinant baculovirus used to infect SF9 cells insect cells Baculovirus protein expression • Used for difficult-to-express proteins in bacteria • Also used for modified proteins • Allows for scale up of expression • Low level of risk: virus is restricted to specific insect cell Left: recombinant insect larva expressing a protein that produces red color Right: an uninfected larva Baculovirus systems Advantages Disadvantages • Can express large proteins (>50 kD) • Grow very slowly (10-12 • Correct glycosylation & signal peptide removal • Cell culture is only • Has chaperonins to help fold “tough” proteins • Set-up is time consuming, • Very high yields, cheap days for set-up) sustainable for 4-5 days not as simple as yeast Mammalian cell line expression • Sometimes required for difficult-toexpress proteins • most eukaryotic vectors based on DNA or RNA viral genomes e.g., human adenoviruses and retroviruses, SV40 • Cells typically derived from CHO cell line ➢ Infection with virus may be stable; may also be transient transfection • Selection must occur for producing large scale amounts of protein Mammalian expression systems Vectors usually use SV-40 virus, CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene Mammalian protein expression • Gene initially cloned and plasmid propagated in bacterial cells • Mammalian cells transformed by electroporation • Gene integrates into random locations w/in diff. chromosomes • Multiple rounds of growth, selection using methotrexate to select for cells with highest expression, integration of DHFR and the gene of interest Methotrexate (MTX) selection Foreign gene expressed in high level in MTX – resistant cells Mammalian systems Disadvantages • Selection takes time (weeks for set-up) • Cell culture is only sustainable for limited period of time • Set-up is very time consuming, costly • Modest yields Advantages • Can express large proteins (>50 kD) • Correct glycosylation & signal peptide removal, generates authentic proteins • Has chaperonins to help fold “tough” ptns Generally used to test the function of a protein in vivo, rather than to produce a protein in large amounts Expression system selection Choice depends on size, character of protein ❑Large proteins (>100 kD)? ❑Small proteins (<30 kD)? ❑Glycosylation essential? High yields, low cost? ❑Post-translational modifications essential? Expression system selection Choice depends on size, character of protein ❑Large proteins (>100 kD)? Choose eukaryote ❑Small proteins (<30 kD)? ❑Glycosylation essential? ❑High yields, low cost? ❑Post-translational modifications essential? Expression system selection Choice depends on size, character of protein ❑Large proteins (>100 kD)? Choose eukaryote ❑Small proteins (<30 kD)? Choose prokaryote ❑Glycosylation essential? ❑High yields, low cost? ❑Post-translational modifications essential? Expression system selection Choice depends on size, character of protein ❑Large proteins (>100 kD)? Choose eukaryote ❑Small proteins (<30 kD)? Choose prokaryote ❑Glycosylation essential? Choose baculovirus or mammalian cell culture ❑High yields, low cost? ❑Post-translational modifications essential? Expression system selection Choice depends on size, character of protein ❑Large proteins (>100 kD)? Choose eukaryote ❑Small proteins (<30 kD)? Choose prokaryote ❑Glycosylation essential? Choose baculovirus or mammalian cell culture ❑High yields, low cost? Choose E. coli ❑Post-translational modifications essential? Expression system selection Choice depends on size, character of protein ❑Large proteins (>100 kD)? Choose eukaryote ❑Small proteins (<30 kD)? Choose prokaryote ❑Glycosylation essential? Choose baculovirus or mammalian cell culture ❑High yields, low cost? Choose E. coli ❑Post-translational modifications essential? Choose yeast, baculovirus or other eukaryote Biomanufacturing: production of pharmaceutical proteins using genetically engineered cells Scaling up of the protein purification process developed during research and development PROTEIN USED IN THE TREATMENT OF Cell Production Insulin Human growth hormone Granulocyte colony stimulating factor Erythropoietin Tissue plasminogen activator Hepatitis B virus vaccine Human papillomavirus vaccine Diabetes Growth disorders Cancers E. coli E. coli E. Coli Anemia Heart attack Vaccination Vaccination CHO cells CHO cells Yeast Yeast Price per gram Recombinant proteins Bovine Growth Hormone $14 Gold $48 Insulin $60 Growth Hormone $227,000 Granulocyte Colony Stimulating Factor $1,357,000 *Prices in 2011 US Dollars The wave of the future: synthetic biology for protein production The wave of the future: synthetic biology for protein production Synthetic proteins • Directed evolution of enzymes • Phage display