ODODODODODODO □ ODODODODODOD ODODODODODODO DODODODODODOD O D O D O ^ ~ ^ O D O D O D O D ^^1J\^V V D O D ODODOu^uODODO DODODODODODOD ODODODODODODO Kód předmětu: BÍ8980 MASARYKOVA UNIVERZITA Protein expression and purification • V. Protein expression Lubomír Janda, Blanka Pekarové and Radka Dopitová Tento projekt je spolufinancován Evropským sociálním fondem a státním rozpočtem České republiky. OP Vzdělávání pro konkurenceschopnost EVROPSKÁ UNIE « NVESTICE I ) ROZVOJE VZDĚLÁVÁNI Název prezentace v zápatí 1 V. Protein expression 5.1. Designing experiments for high-throughput protein expression High-throughput platform requires: • Automation Miniaturization Quantitative management tools (to identify trends and relationships) An ill-defined experiment will often produce ambiguous results and fail to reach any conclusion. Analysis of quantitative response allows the experimenter to optimize conditions critical to production of a soluble protein. Performing one-factor-at-a-time experiments raises the risk of locating a local maximum (missing the actual best conditions). A: One factor at a time B: Fractional factorial C: Full factorial D: Response surface model (Box-Behnken design for three factors) V. Protein expression 5.1. Designing experiments for high-throi Factors affecting expression: • Construct Expression system and vector Cell line (host strain) Temperature and time • Media • Additives Full factorial design (16 conditions per construct): • three continuous factors (temperature, time, IPTG concentration) • one categorical (host strain) V. Protein expression 5.1. Designing experiments for high-throughput protein expression Response surface model: • fine-tunes the conditions capable to identify minimum or maximum I 1 J 1 I 25 30 35 Temp (°C) r 40 21 D 37 Design of experiment is merely a statistical tool, a means to an end. It does not guarantee success and cannot replace technical expertise or creativity in experimental work. 5.2. Approaches for efficient protein production V. Protein expression I. Genetic approach x protein knowledgebase (biochemical approach) II. Expression density x functional activity III. Expression system x medium engineering IV. Troubles with removing tag fusion proteins x less convenient purification with classical chromatography ^^Bin expression ^^^^H 5.2. Approaches for efficient protein production 5.2.1. Genetic approach x protein knowledgebase ATGGGCGGCATccACAGGGTGAACAGATGTACCGGAGGGTGTATCGTCTGCATGAGCGCCTGGTAGCCATCCGCACTGAGTACAACCTCC GGCTGAAGGCAGGAGTGGGTGCCCCTGTGACCCAGGTGACCCTGCAGAGTACACAGAGGCGCCCAGAGCTAGAGGACTCCACACTGCGCT ACCTGCAAGACCTGCTGGCCTGGGTAGAGGAGAACCAGCGTCGAATAGACAGTGCTGAGTGGGGCGTGGACTTGCCCAGTGTGGAGGCCC AGCTGGGCAGCCACCGAGGCATGCATCAGTCTATAGAGGAATTTCGGGCCAAGATCGAGCGGGCTCGGAATGATGAGAGCCAGCTCTCCC CTGCCACCCGGGGTGCCTACCGGGACTGCCTAGGTCGCCTAGACCTGCAGTATGCAAAGCTGCTGAACTCCTCCAAGGCCCGCCTCCGGT CCCTGGAG^^^^^^^A ^^^^^^^^^^^^riririAririrpA ririA 7\pp7\pnTn7\nnp^nTP7\ att^ Ar^ a a ana ana.anna an^rn^QCTTTGATT 'io j 5= r 3 ?S .i; GGAGTGACC ^ y y O cj ^ ES,y ^ y ^AAATCAAGG AGATCCAG^ O rh X ^ ;j U-i 33 ^ S ^ S ?S TGAAGCCAC , o 5? ^ * ^ 2,*^ ^ ■ S ^.cTGTGCACA AGGGTGACC BB '3 . jj ^ 1 ■ PC • jTGCCTTCTG TGTGCTTTC £ ^ cL ! § IL ■§ ^CACTGTGGC ACCAGCTTC ... UTAGTCACGT 2 ^ ><, era TCCGCACAC I 1 \^ I 1 ^4^Vi^ I HAGGATGCCG GTGGCTTTG - 1 ^.GCCTGGAGC AGGGTGAGC DGVRANELQLRWQEYRELVLLLLQWIRHHTAAFEERKFPSSFEEIEILWCQFLKFKETELPAKEADKNRSKVIYQSLEGAVQAGQLK IPPGYHPLDVEKEWGKLHVAILEREKQLRSEFERLECLQRIVSKLQMEAGLCEEQLNQADALLQSDIRLLASGKVAQRAGEVERDLD KADGMIRLLFNDVQTLKDGRHPQGEQMYRRVYRLHERLVAIRTEYNLRLKAGVGAPVTQVTLQSTQRRPELEDSTLRYLQDLLAWVE ENQRRIDSAEWGVDLPSVEAQLGSHRGMHQSIEEFRAKIERARNDESQLSPATRGAYRDCLGRLDLQYAKLLNSSKARLRSLESLHG LQLCCCIEAHLKENTAYFQFFSDVREAEEQLQKLQETLRRKYSCDRTITVTRLEDLLQDAQDEKEQLNEYKGHLSGLAKRAKAIVQL VEECQKFAKQYINAIKDYELQLITYKAQLEPVASPAKKPKVQSGSESVIQEYVDLRTRYSELTTLTSQYIKFISETLRRMEEEE V. Protein expression 5.2. Approaches for efficient protein production 5.2.1. Genetic approach x protein knowledgebase V. Protein expression 5.2. Approaches for efficient protein production 5.2.1. Genetic approach x protein knowledgebase Plectin N-terminal domain rod C-terminal domain Tau1 Tau2 Tau3 MAP2/1 MAP2/2 MAP2/3 Pledc 848-905 M1 domain_ SH3 1392 T E N L K H Q P G G G L G N I H H K P G G G L D N I T H V P G G G T D N I K Y Q P K G G L K N I R H R P G G G L D A H H V P G G G K T R S R R S G G G 2518 4589 Kd of Plectin (Ex 1 -24) for actin 320 nM Kd of Plectin (R5) for vimentin (IF) 100 nM Kd of Plectin (Ex 2-8) for integrin beta 4 170 nM 2739 3067 IFBD RDs Linker Module 6 25 5 PlecMouse 'AAAQSSKGYYSPYSVSGSGSTAGSRTGSRTGSRAGSRRGSFDATGSGFSMTFSSSSYSSSGYGRRYASGPSASLGGPESAVA1 PlecHamst AAAQSSKGYYSPYSVSGSGSTTGSRTGSRTG5RAG5RRGSFDATGSGFSMTFSSSSYS5SGYGRRYASGPPA5LGGPESAVA PlecHuman AAAQSTKGYYSPYSVSGSGSTTGSRTGSRTGSRAGSRRGSFDATGSGFSMTFSSSSYSSSGYGRRYASSPPASLGGPESAVA (Ex 2-8) 25uM Kd for microtubules in case of MAP2 1-3 uM ^^Kin expression 5.2. Approaches for efficient protein production 5.2.1. Genetic approach x protein knowledgebase C-terminal domain ESAVA* V. Protein expression 5.2. Approaches for efficient protein production 5.2.1. Genetic approach x protein knowledgebase Spectrin 978 Actinin DM 834 Itk Tyr kinase 167 Envoplakin II 404 Actinin CE 847 Periplakin H 391 Eakapo DH 793 Kakapo AG 941 Desmoplakin H 536 MACF 8S3 Dystonin H 877 Plectin H 931 E PRD domain_ l Bt HednrrsfqeGeetlI q rhn HseqHlh l tu q r g e r^h h h i e l e y r r e t ml e | lnerrq kvnrq l e q re q bvn r q c t leHrnHdyrsne0ii leHrnGdhvlestls L F SH3 domain PRD G L F RNBD ] lets rn ai VUG a Intramolecular SH3-Iigand association PK t Accessible pTyr binding site Bidemate SH2/SH3 ligand SH2 ŤH K PH ) b jEKNDVLT j D E G e V H L R C D e e Y L R G e R C t EA GD D V S R G Y S Y t eH e t V t D EH e t C t HE GD e C I CKNDEC YED D e C H E G D Q C Q| L S S I If D TBI |E S eBnD D TJE lIsI E I H TiJI IEDHADPYTI LDNSDLIEI Q E N N G E SI L D H S G R V El L D T S G R V El lEDNNERSEI EDNSQRTEI I ANN S H R A EE VGPAQPSHI eaddhqg f v p a v re l a| red n g v e g f v p an r e v e q d en gh e g y aI S S v e e S q g p g g e t e S a p a a c tu i p a rd i S g a e gq v p S v f rB hd S a 1 n e l i aB c f v i r t ae g q e g p i p c l l l e t S e g v e g S vh c l l l t g p g g vd h l v p S g l i i i S p t gn e ahv p S c f l i i S p t gn e a vv p S c f t v l S g S S S e a a v p S c f l v a) Model of observed intramolecular interaction showing the observed interaction between the Itk proline-rich region and SH3 domain. b) Model of the opening of the intramolecular complex by interaction with bidentate ligand for the Itk SH3 and SH2 domains. (Andreotti et al., Nature 1996) Accessible proline-rich site ^^^Mxpression I 5.2. Approaches for efficient protein production 5.2.1. Genetic approach x protein knowledgebase SH3 domain of plectin with surrounding proline rich regions (Sarc homology domain soluble in citrate buffer of pH 3.5) Average Mw: 8,732.3 Da (7 parallel measurements) RSD: 0.02% Theoretical pI/Mw (average) for the protein sequence Theoretical pI/Mw: 7.78 / 8,726.11 mefKAIVQLKPRNPAHPVRGHVPLIAVCDYKQVEVTVHKGD QCQLVGPAQPSHWKVLSGSSSEAAVPSVCFLVPPPNQEf 2 0 0 15 0 10 0 b 0 6 0 0 0 7 0 0 0 8 0 0 0 9 0 0 0 0000 m / V. Protein expression 5.2. Approaches for efficient protein production 5.2.2. Expression density x functional activity Expression and purification of plectin's ABD (Actin Binding Domain) in three isoforms. Julius Kos tan V. Protein expression 5.2. Approaches for efficient protein production 5.2.2. Expression density x functional activity Maize recombinant ß-glucosidase produced in E. coli. Cultivation condition Yield (mg) Specific activity (nkat/mg) /(total activity nkat) LB medium 380 1.9 (966 nkat) TB medium - pH 6 230 3.8 (874 nkat) TB medium — pH 7 230 4.2!*) (966) nkat) TB medium —pH 8 410 2.8 (1,148 nkat) Additive of cellobiose (LB medium) 400 2.7! (1,080 nkat) Radka Fohlerov Result: TB medium (pH 7.0) supplemented by cellobiose shows 3.1 x higher ß-glucosidase specific activity than in common LB medium. ^^Bin expression I I 5.2. Approaches for efficient protein production 5.2.2. Expression density x functional activity The cytolinker protein: plectin 4Í4 Plectin R5 ^^Bin expression I I 5.2. Approaches for efficient protein production 5.2.2. Expression density x functional activity Converted pET 15b + IF binding domain of plectin • R5 d. plectin (pH 7.9) • R5 d. plectin (pH 7.9, urea, dialysis) • R5 d. plectin (pH 7.9, urea, refolding HR) • R5 d. plectin (pH 11, purification pH 9.0) Co-sedimentation - functional test on protein activity A kDa ^_ <ď s£ S P S P S P B ^95%) 45 25 R4 Kamaran Abdoulrahman 1. R5wt oxidased form + DTT 2. R5wt oxidased form - DTT V. Protein expression 5.2. Approaches for efficient protein production 5.2.2. Expression density x functional activity Expression and purification of plectin's ABD (actin binding domain) in three isoforms. Julius Kos tan V. Protein expression 5.2. Approaches for efficient protein production 5.2.2. Expression density x functional activity Monoclinic crystals of plectin ABD Precipitant solution: 0.1 M TRIS buffer pH 8.5 10% PEG 4000 2% dioxane Ľubica Urbaníková Space group P2i 2 molecules in asym. unit Ľubica Urbaníková ^^Bin expression I I 5.2. Approaches for efficient protein production 5.2.2. Expression density x functional activity Orthorhombic crystals of plectin ABD Precipitant solution: 0.1 M Cacodylate buffer pH 6.5 6-8% PEG 8000 0.2 M Ca acetate 2% dioxane Space group P212121 1 molecule in asym. unit 2.0 A resolution V. Protein expression 1nlv: S.M.Vorobiev, B. Strokopytov, DG. Drubin, C. Frieden, S. Ono, J. Condeelis, P.A. Rubenstein, S.C. Almo. The structure of non-vertebrate actin: Implications for the ATP hydrolytic mechanism (2003). ProcNatlAcadSci. USA 100:5760-5765. 1 rgi: L.D.Burtnick, D. Urosev, E. Irobi, K. Narayan, R.C. Robinson (2004). Structure of the N-terminal half of gelsolin bound to actin: roles in severing, apoptosis and FAF. EMBOJ. 23:2713-2722. 1izn: A.Yamashita, K. Maeda, Y. Maeda (2003). Crystal structure of CapZ: structural basis for actin filament barbed end capping. EMBOJ. 22:1529-1538. 1sh5: J. Sevcik, L. Urbanikova, J. Kostan, L. Janda, G. Wiche (2004). Actin-binding domain of mouse plectin: crystal structure and binding to vimentin. Eur.J.Biochem. 271:8731884. Actin July 2008 PROTEIN DATA BANK The cytoskeleton is an intracellular maze of filaments thai supports and shapes the fell. The most plentiful type of filament is composed of octin, shown here in blue. The cyloskelelon, however, is not a static structure, since h must respond !o 1 he changing needs of the cell. The proteins shown here help to reshape the cyloskelelon by assembling or disassembling actin filaments as necessary. A molecule of ATP, which h bound inside each actin molecule, is important in this process. When r is hydrolyzed to ADp the filament becomes unstable and falls npnr:. Gelsolin breaks down actin filaments by assisting the hydrolysis of ATP and blocking the sites of interaction with other actin proteins. Two different fragments of gelsolin are shown in Inlv and 1 rgi bound to actin. The protein CapZ forms a cap on the odin filaments shown in tizn, which limits assem The protein I the proper orientation which starts the process of filament growth. One domain of formin is shown bound to octin in 1 y64 Plectin links neighboring actin filaments into higher order structures. The aclin-bmding domain is shown in lsh5. V. Protein expression 5.2. Approaches for efficient protein production 5.2.3. Expression system x medium engineering Examples of E. coli expression systems and web pages for further information Vector Promoter/ system induction method Special host strains required Protein tag Source (website) * tac/IPTGor77IPTG Yes 77IPTG Yes tac/IPTG No araBAD Yes Pi/trp Yes T7IPTG Yes Pitet/anhydrotetracycline No T7 IPTG Yes tac/IPTG Yes T5/IPTG Yes/TOPP T7/IPTG Yes Biotin binding domain www.promega.com His6,T7gene http://www.merckbiosciences.co.uk GST www.amershambiosciences.com His6, GFP www.invitrogen.com www.clontech.com tac/IPTG Yes His6, T7 His6 Chitin binding domain www.neb.com Maltose binding domain His6 www.qiagen.com Calmodulin binding www.stratagene.com peptide www.sigmaaldrich.com ^^Kin expression 5.2. Approaches for efficient protein production 5.2.3. Expression system x medium engineering pET32a::AHP1 pET32a::AHP5 Ava 1(158) Xho 1(158) Eag 1(166} Not 1(166} Hind 111(173) Sal 1(179) Sac 1(190} EcoR 1(192) BamH 1(198J EcoR V(2oe) Nco 1(212) Sea 1(4995) PVU 1(4885) Pst 1(4760} Bsa 1(4576) Eam1105 1(4357) AlwN 1(4038) AHP - phosphotransfer protein in cytokinin signalling pathway of A. thaliana BssH 11(1932} Hpa 1(2027) BspLU11 1(3622) Sap 1(3506) 8S11107 ((3393) Tth 111 1(3367) BspG 1(3148} PshA 1(2366) Psp5 11(2628} pRSETB::AHP1 pRSETB::AHP5 S O o ~ ~ a t G jSIS' BS ATG 6xHts Xpress™ Epitope EK pRSET A,B,C 2.9 kb AHP5 ^^Kin expression 5.2. Approaches for efficient protein production 5.2.3. Expression system x medium engineering 5.2.3.1. Temperature Expression system Plasmid pRSETB Temperature (V) growth/induction Soluble form (%) Insoluble form (%) Soluble form (%) Insoluble form (%) 22CC/22CC C§2^ 38% /71°/o\ 29% 37°C/22CC 0% 100% 82% 18% 37°C/28CC 0% 100% 8% 92% Plasmid pET32a+ pRSETB Temperature (°C) growth/induction Soluble form (%) Insoluble form (%) Soluble form (%) Insoluble form (%) 22CCC/22CC 22% 76% 24% 37CCC/22CC 67% 33% 81% 19% 37CCC/28CC 61% 39% 81% 19% Radka Fohlerová V. Protein expression 5.2. Approaches for efficient protein production 5.2.3. Expression system x medium engineering 5.2.3.1. Temperature Production of soluble AHP proteins using E. coli expression vector pRSET at different temperatures (%) Temperature (V) growth/induction AHP1 AHP2 AHP3 AHP4 AHP5 AHP6 8% (85% 100% 0% 76% 0% 37°C/22CC 82% 73% 100% 0% 81% 51% 71% 78% 100% 30% 81% 73% Radka Fohlerová ^^Kin expression 5.2. Approaches for efficient protein production 5.2.3. Expression system x medium engineering 5.2.3.2. Medium pH Production receiver domain of plant histidine kinase AHK4 in E. coli by pET161DEST pH Soluble fraction 6.0 35% 7.0 89% 8.0 100% • Disintegrate E. coli in native buffer. • Denaturated crude extract by chaotropic compounds (urea). • Load SDS-PAGE. • Scan the gel after staining and subsequent de-staining. • Evaluate differences between signals from protein denaturated by chaotropic compounds and protein signal from native buffer. ^^Kin expression 5.2. Approaches for efficient protein production 5.2.3. Expression system x medium engineering Met 944 His 112: pDEST17::CKI1ex1 - 371 AA, Mw = 42 kDa pDEST17::CKI1ex2 - 419 AA, Mw = 47 kDa V. Protein expression 5.2. Approaches for efficient protein production 5.2.3. Expression system x medium engineering Growth temperature 37^, expression 28^ Growth and expression 25°C 1 2 3456 7 8 9 1: BL21 — 1 h after induction = OD 0.5 2: 4 h after induction = OD 0 5 3: H h after induction = QD 2 I 4: 4 h after induction = OD 2 -5i--- 6: 7: 8: 9 C43 — 1 h after induction = OD 0.5 4 h after induction = OD 0.5 1 h after induction = OD 2 4 h after induction = OD 2 S: 14-66 kDa 1: 2: 3: 4: 5: 6: 7: 8: 9: BL 21 before induction 1 h after induction = OD 0.5 3 h after induction = OD 0.5 2 h after induction = OD 2 C43 before induction 1 h after induction = OD 0.5 3 h after induction = OD 0.5 2 h after induction = OD 2 S: 14-66 kDa 1 2 |~3 j 4 5 6 7 8 9 Petra Borkovcovä s ^^Kin expression 5.2. Approaches for efficient protein production 5.2.3. Expression system x medium engineering Citrate b., pH 3.6, Triton X-100 Tris b., dH 7.9, Triton X-100 Tris b., pH 7.9, Triton X-100 Tris b., pH 7.9, CTAB Tris b., pH 7.9, NONIDET P-40 Tris b., pH 7.9, SDS Tris b., pH 7.9 V. Protein expression 5.2. Approaches for efficient protein production 5.2.3. Expression system x medium engineering A. E. coli BL21(DE3) Arctica B. E. coli BL21 (DE3)RjL A. T . ~. . B. _ . Tris Glycine Tris Glycine CAPS A- pH 11 pH 11.5 B-pH 11 pH 11.5 S P S P SP Tween Western blot detected by poly-His antibodies SP S P S P S P S P NP40 Deoxycholate P S P S P Severine Jansen V. Protein expression 5.2. Approaches for efficient protein production 5.2.3. Expression system x medium engineering 5.2.3.3. Buffer for disintegration (pH) LTP-2 (non-specific lipid transporting protein from wheat) pH medium pH 6.0 pH 7.0 supernatant Buffer for disintegration (pH) T t Glycine buffer pH 10.6 Phosphate buffer pH 7.2 pH 6.0 pH 7.0 pellet Kateřina Sikorová V. Protein expression 5.2. Approaches for efficient protein production 5.2.4. Troubles with removing tag fusion protein x less convenient purification with column N-terminal amino acids which reduce protein stability Arg, Lys, Phe, Leu, Trp and Tyr Tobias et al., 1991, Science Amino acids in penultimate position (second behind N-terminal methionine) enhancing protein stability. • His, Gln, Glu, Phe, Met, Lys, Tyr, Trp, Arg - Hirel et. al., 1989, PNAS and Lathrop et al. 1992 - Liao et al., 2004, Protein Science V. Protein expression 5.2. Approaches for efficient protein production 5.2.4. Troubles with removing tag fusion protein x less convenient purification with column Control of protease cleavage sites in fusion proteins Pro-Arg/Gly Pro-Lys/Leu Ala-Arg/Gly Gly-Lys/Ala Leu-Glu-Val-Leu-Phe-Gln/Gly-Pro lle-Glu-Gly-Arg/X Asp-Asp-Asp-Asp-Lys/X AHP5 V. Protein expression 5.2. Approaches for efficient protein production 5.2.5. Maximizing target protein recovery • Protein expression • Take advantage of protein knowledgebase for construction of expression vector Protein purification • Medium engineering • protein induction at different temperature • medium pH • additives • buffer options for disintegration • degas all the buffers • Test on protein activity • Encourage protein purification without tag sequences V. Protein expression 5.3. Expression system 5.3.1. E. coli expression system Advantages and Disadvantages of E. coli • Ease of gene manipulation • Availability of reagents • Easy of producing quantities of protein • Speed • Low cost • Adaptability of the system • Formation of insoluble inclusion bodies • Size of the protein • Post-translational modification V. Protein expression 5.3. Expression system 5.3.1. E. coli expression system Plectin N-terminal domain 1 315 848-905 ABD MTBD /r\ SH3 rod C-terminal domain 1392 2518 2739 30ů7 4589 PESAVA* KRNPAHPVRGHVP Size of the protein (LI 1c-30 MEFHMSGEDS ... TLRRMEEEEF pl/Mw: 6.37 / 160,602.22 lc-24 MEFHMSGEDS ... CISELKDIEF : pl/Mw: 6.38 / 119,799.86 RDs Linker Module Soluble form Inclusion bodies Nt . ld - f - ť At. b.d. d i f , f N-terminal domain of mouse plectin Actin binding domain of mouse plectin 5.3.2. Baculovirus protein expression system V. Protein expression • HT-bacmid propagation • HT-suspension-based insect cell transfection • Methods of recombinant viral titer determination GFP co-expression, titration assay using Alamarblue, Cedex cell counter • HT-miniaturized deep-well block insect cell expression • Transient insect cell expression V. Protein expression 5.3. Expression system 5.3.2. Baculovirus protein expression system Overview on commercially available baculovirus expression systems Methodology Transfer of foreign Baculovirus for chining gene into Selection/ expression Compatible la reign gene into Baculovirus Recombination kits and vendors transfer vectors transfer vector genome efficiency Jiad?AK1H (Clofiiech) &a*cd on homologous recombination at polyhedriirt Ickjus Ligase dcpcndenr Homologous recombination in insect oelfe 590% Bac-io-Bac™ (Inviíifůgřfl) Haw:J on site' specific transposition Ligase-depciident^ Gateways adapted Sirc-spccinc transposition in bactcriaJ cells Seiet tion of recombinants by blue-white selection on agar plates Rased on site-specific recom.-binatrion Gateway"" adapted Site-specific recombination in Epi>endor(~ cube Antibiotic selection of transfectauts in insect cells flashBac1- (OET/ NextÜen Sciences) Based on homologous recom-binatiou at potyhedrin iocus Ligasc dcpci ■ Honiologous recombination in insect cells BaeVector™ L000T 2000x30ÜO Based on homologous recombination at polyhcdrin locus Ligase dependent Homologous recombi nation in insect cells BaculoGold'" (Cloentcch) Based on homologous recombination ac polyhcdrin locus Ligase dependent Homologous recombination in insect cells niarnondßac'* (Sigma-ALdrich) Isased on EiLMnologou? ar polyhcdrin locus Liaise dependent Homologous recombination in insect cells V. Protein expression 5.3. Expression system 5.3.3. Cell-free protein expression system Simple open system which influences: • Protein folding • Disulfide bond formation • Incorporation of unnatural amino acids • Protein stability • Expression of toxic proteins Use the machinery of E. coli S30 V. Protein expression 5.3. Expression system 5.3.4. Transient protein expression in tobacco leaves An Agrobacterium-\nedi\ateti transient expression assay has been described for in vivo analysis of constitutive or inducible gene expression in Arabidopsis plants. • Plant number: ca 30 • Weight of tobacco leaves: 7-10 g • Number of tobacco leaves: 12-15 • Total: ca 3.5 kg~12-15 g protein~120-150 mg scFv 31,0 1 scFvx DHZR in Tobacco 17 «8 B9 ■ 10 Bll 12 B16 18 66 9|2 50,2 48,o 49,2 " 47,8 ■ ■■■■■ I 8 9 10 11 12 16 18