RECOMBINANT DNA TECHNIQUES and PROTEIN ENGINEERING

1. RECOMBINANT DNA TECHNIQUESandPROTEIN ENGINEERING Expression systems Genetically modified organisms

2. E. coli expression systems • PROMOTERS • trp-lac hybrid promoters (tac, trc) trp –35 region +lacUV5–10 region (Pribnow-box) • TATGTT(WT) • TATAAT(2,5x stronger, no CRP regulation, GA –66) • „linker” –10, –35 between 16 bp (tac), 17 bp (trc) • overlapping lac operator sequence • repressed by lac repressor • induction: IPTG (~50x)

3. Other promoters  PL promoter from bacteriophage l induction: 30oC42oC temperature sensitive  repressor) host: defective  lysogen (cI857) e.g.: pAS vectors araBAD arabinose operon AraC regulated by arabinose(positive)and glucose (negative) induction: arabinose (dose dependent)( ~1200x) e.g. pBAD vectors (Invitrogen)

4. Other promoters T7 RNA polymerase promoter (10) E. coli RNA polymerse can not use needs the presence of T7 RNA polymerase gene infection by phage transformation with a compatible vector containing the gene (pGP1-2) prophage in host chromosome (DE3 lysogen)regulated by lac promoter/operator induction: IPTG host: BL21(DE3) (lysogen, lon-, ompT-) pLys plasmid: codes T7 lysozim gene (inhibitor of T7 pol) pl. pT7-7 pET vectors (Novagen)

5. System with two plasmids pT7/7 – pGP1-2

6. pET vectors

11. Secretion in E.coli Disulfide bond containing proteins phoA alkaline phosphatase periplasmic secretion signal peptide fusion, in vivocleavage reguláció: phoB (pozitive), phoR (negative) induction: phosphate depletion e.g.: pTrap (trypsinogen expression) pelB signal („leader”) peptide

12. Fusionsystems Goal: to enhance solubility, stability and purification of the recombinant proteins glutathion-S-transferase from Schistosoma japonicum (26 kd) easy isolation (glutathion-agarose) pGEX vectors Maltose-binding protein fehérje E. coli malE gene isolation on maltose affinity column pMAL vectors thioredoxin E. coli trxA gene (ribonucleotide-reductase reducing enzyme) „super” soluble, thermostable (80oC) small protein(16,7 kD) pTrxFus

13. GFP (green fluorescent protein) Aequoria victoria jellyfish superstable protein (238 a.a.) chromophor: Ser(Thr)-Tyr-Glyp-hydroxybenzylideneimidazolidin (auto catalytic maturation) the expressed fusion protein is fluorescent in many different cells, even in whole organisms in vivo marker gene

14. IMPACT™ (Intein Mediated Purification with an Affinity Chitin-binding Tag)

15. AFFINITY TAG FUSIONS His-tag 6xHis at the N- or C- terminus Ni-affinity resin (Ni2+-nitrylotri acetic acid) pl. pET15 (N), pET20 (C) T7-tag T7 gene 1 leader sequence (11 a.a.) translation enhancer Strap-tag C-terminal AWRHPQFGG (affinity for streptavidin) epitope-tags (good monoclonal antibodies availeble) FLAG-peptide (DYKDDDDK), c-myc-tag

16. Clevage of fusion protein: thrombin LVPRGS factor Xa I(D/E)GR enterokinaseDDDK TEV protease ENLYFQ↓G preScission LEVLFQ↓GP BrCN; Met hydroxylamine Asn-Gly

17. Problems, solutions Codon usage: different between eukaryots and E.coli Arg (AGG,AGA) argU (tRNA gene) coexpression Rosetta host tRNAs for 8 rare codons Distance between SD sequence (…AGGA…) and first ATG 5-11 bp Internal SD/terminator seq., strong secondary structure in insertsequence analysis, mutations mRNA stability (30 sec-20 min) ompA 5’-UTR Termination of translation UAA, UAAU Instability of expressed proteins: degradation, misfolding protease deficient hosts: lon-, opmT-, clp-, fts- chaperonin coexpression (DnaK-DnaJ, GroEL-GroES) cold induction (cold shock promoter; cspA) fusion protein

18. Problems, solutions inclusion body formation: easy to isolate, protection against proteases in vitrorefolding periplasmatic expression NO POST-TRANSLATIONAL MODIFICATION Coexpression with eukariotic enzyme

19. Yeast expression systems S. cerevisiae vectors („shuttle”): YEp, YCp, YIp selection markers: LEU2, TRP1, URA3, HIS3 (auxotroph host) promoters: strenth regulation PGK, GAPD++++weak induction by glucose GAL1+++stronginduction by galactose ADH2++repression by glucose PHO5++repression by phosphate Hybrids, e.g. ADH/GAPD terminators: PGK, GAPD, CYC1 termination of transcription, processing, polyadenylation

20. secretion systems: -factor („mating”feromon), prepro sequence cleavage: signal peptidase KEX2 protease pl. pYES2 (5,9 kb) (Invitrogen) pYX family (Novagen)

21. Pichia pastoris Methylotroph yeast (consumes methyl alcohol) superexpression , g/l quick, simple, cheap promoter: alcohol-oxydase (AOX1) induction: methanol transfer vectors („shuttle”) (Invitrogen) insertion of vector into the chromosome by homologous recombination selection: His+, zeocin

24. Mammalian expression system Cell cultures transient (12-80 hours) or stable (clon selection) modules in the vectors E. coli replicon (shuttle) mammalian promoter/enhancer (RNA pol II) transciption termination and polyadenylation signal (AATAAA) splicing signal (intron) translation initiation (Kozak sequence: CCA/GCCAUG) marker gene (with mammalian promoter) Cell cultures monolayer, suspension primer, „established” (immortalised) culture NIH-3T3, HeLa, COS, L6, HEK-293, … require vitamins, essential amino acids, serum (growth factors) antibiotics EXPENSIVE, SENSITIVE

25. transfection : DNA bound to DEAE-dextrán, Ca-foszfát lipofection (cationic liposome), electroporation, selection : TK- (thyimidin-kinase deficient) cell + HSV TK gene cotransfection dTMP „salvage” synthesis HAT medium (hypoxantin, aminopterin, thymin) DHFR- (dihydrofolate-reductase deficient cell pl. CHO) + DHFR+ vektor + metotrexát (antifolate)  amplification (high copy number) antibiotics resistance neomycin (G418, Geneticin), hygromicin, zeocin, blasticidin

26. In vitro protein production: RTS Technologies (Roche) • coupled in vitro transcription and translation from DNA!!! • Prokaryotic (E.coli) or eukaryotic (wheat germ) extract supplies ribosomes, tRNAs , protein factors and enzymes • CECF (Continuous Exchange Cell-Free) technology

27. In vitro protein production: RTS Technologies (Roche) • E. coli

28. In vitro protein production: RTS Technologies (Roche) • production of linear template by overlap extension

29. In vitro protein production: RTS Technologies (Roche) • wheat germ

30. In vitro protein production: RTS Technologies (Roche) Monobiotinylated tag, BirA: biotin protein ligase recognition sequence N-terminal tag sequence: MSGLNDIFEAQKIEWHE C-terminal tag sequence: LERAPGGLNDIFEAQKIEWHE

31. In vitro protein production: RTS Technologies (Roche) RTS 100: ~ 100 mg/ml/24 hours RTS 500: 1-5 mg/ml/24 hours

32. GMO-s • Organisms: • Bacteria • Yeast, Pichia • Plants • Animals • Introduction of DNA constructs: • Transformation: plasmids • Transfection: phages, viruses • Electropotation • Gene gun • Microinjection • Basic methods • Gene knockout • Gene knock in • Post transcriptional gene silencing

33. Gene knock-out Negative selection: HSV thymidilate kinase + ganciclovir

34. Geneknock in

35. Transformation of plants • Plant cells are totipotent • Can be in infected by Agrobacterium • Foreign DNA incorporated into Ti plasmid • Selection by antibiotics • After infection foreign DNA integrates into genom • Regeneration plants from single cell

36. The Gene gun

37. Discovery of PTGS • First discovered in plants • (R. Jorgensen, 1990) • When Jorgensen introduced a re-engineered gene into petunia that had a lot of homology with an endogenous petunia gene, both genes became suppressed! • Also called Co-suppression • Suppression was mostly due to increased degradation of the mRNAs (from the endogenous and introduced genes)

38. RNA interference

39. Transgenic mice – ES technique

41. Thanks for your patience