ODODODODOöODO □ OüODOüOüOüOü ODODODODOÖODO □ OP.ODOP.OnOP.OD O □ O □ o Z. o □ O □ O Kód předmětu: C8980 □ o p -oj JD-t\,}/>n ° D o r/ÄV n o I 1MJ * MASARYKOVA UNIVERZITA □ ocíQ^NA^0 ° OPODOu^uOOOPO □ OüODOüOüOüOü ODODODODODODO Protein expression and purification • V. Protein expression Lubomír Janda, Blanka Pekarové, Radka Dopitová, Jozef Hritz and Adam Norek Tento projekt je spolufinancován Evropským sociálním fondem a státním rozpočtem České republiky. A* OP Vzdělávání pro konkurenceschopnost evropská unie « INVESTICE DO ROZVOJE VZDĚLÁVÁNÍ Protein expression 5.1. Designing experiments for high-throughput protein expression CD H—1 03 CD CL E CD High-throughput platform requires: • Automation • Miniaturization • Quantitative management tools (to identify trends and relationships) Experimental design: if / • 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). I. 15°C/1h/0,1mM IPTG II. 15°C/24h/0,1 mM IPTG III. 15°C//24h/1mM IPTG IV. 15°C/1h/1mM IPTG V. 37°C/1h/0,1 mM IPTG VI. 37°C/24h/0,1 mM IPTG A: One factor at a time B: Fractional factorial C: Full factorial D: Response surface model (Box-Behnken design for three factors) VII. 37°C/24h/1mM IPTG VIII. 37°C/1 h/0,1 mM IPTG 2 Experimental design (commonly referred to as DOE) is a useful complement to multivariate data analysis because it generates "structured" data tables, i.e. data tables that contain an important amount of structured variation. This underlying structure will then be used as a basis for multivariate modeling, which will guarantee stable and robust models. More generally, careful sample selection increases the chances of extracting useful information from the data. When one has the possibility to actively perturb the system (experiment with the variables), these chances become even greater. The critical part is to decide 1) which variables to change, 2) the intervals for this variation, and 3) the pattern of the experimental points. 1) Temperature 2) 15°C-37°C 3) About 1°C;5°C or 10°C Temperature 15°C-37°C~23 culture conditions IPTG 0,1 - 1,0 mM -10 culture conditions Time 1 h-24h -25 culture conditions 23x10x25=5750 culture conditions Protein expression 5.1. Designing experiments for high-throughput protein expression Factors affecting expression: • Construct - Cat • Expression system and vector - Cat • Cell line (host strain) - Cat • Temperature and time - Cont • Media - Cat • Additives - Cat Full factorial design (16 conditions per construct): • three continuous factors (temperature, time, IPTG concentration) • one categorical (host strain) 4 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 3h 18h 1 I 1 I 1 I 25 30 35 Temp (°C) "T 40 J B 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. Microreactor technologies developed at LLNL use micromachining techniques to miniaturize the reactor design. Schematic diagram of integration of nano-UPLC (ultra performance Mixed Sample liquid chromatography) with droplet-based microfluidics. UPLC output Oil Water Organic Front of peak 4 Tail of peak 3 Peak 3 Front of peak 3 ntuition, experience and good judgment 999999996 Protein expression 5.2. Approaches for efficient protein production Genetic approach x protein knowledgebase (biochemical approach) Expression density x functional activity Expression system x medium engineering Troubles with removing tag fusion proteins x less convenient purification with classical chromatography DNA cloning 4.2. The key questions before DNA cloning 4.2.1. DNA-protein analysis 4.2.1.2. Secondary structure prediction www.expasy.ch jpred3 mastdsesetrvksvrtgrkpignpedeqetskpsddeflrgkrvlvvddnfisrkvatg —eeeeee----eeeeeeeeee-----------------.-^-'--eeeeee—hhhhhhhhh KLKKMGVSEVEQCDSGKEALRLVTEGLTQREEpG^vdklpfdyifmdcqmpemdgyeatr hhhh----eeeee—hhhhhhhhhh-------------eeeeee------hhhhhh eirkveksygvrtpiiavsghd^@seearetiqagmdafldkslnqlanv.jreieskrh hhhh---------eeeee^--—hhhhhhhhhh-----e---HHfJHtfTHHHHHHHH--- ................. 4.2.1.3...EK5mains detected by SMART _________ .............. www^pasv.ch SMART ......... ...«*-—— ........ ^vlvvddnfisrkvatgk^kkMgvseveqcdsgkealrlvtegltqreeqgsvdklp fdyifmdcqmpemdg.¥-e^treirkveksygvrtpiiavsghdpgseearetiqagmda fldkslnqlanvi** Confidently predicted domains, repeats, motifs and features: Name Begin End E-value REC 43 171 1.19e26 100 200 V. Protein expression 5.2. Approaches for efficient protein production 5.2.1. Genetic approach x protein knowledgebase Histidine kinase from rij A. thaliana - CKI 1 ~^ TM doména EH Three constructs of receiver domain. pET 28 0.075- 0.050 0.025 1 2 314 5 6 7 8 10 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 315 ABD MTBD /T\- 848-905 M1 domain 1392 Tau1 Tau2 Tau3 MAP2/1 MAP2/2 MAP2/3 Pledc s K I s K C s K I s K s K c A K V W N L SH3 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 I K Y Q P K G G L K N I R H R P G G G L D N A H H V P G G G K T Q R S R R S G G G 2518 4589 2739 3067 ■ a IFBD RDs Linker Module 6 25 5 PlecMouse "AAAQSSKGYYSPYSVSGSGSTAGSRTGSRTGSRAGSRRGSFDATGSGFSMTFSSSSYSSSGYGRRYASGPSASLGGPESAVA1 PlecHamst AAAQSSKGYYSPYSVSGSGSTTGSRTGSRTGSRAGSRRGSFDATGSGFSMTFSSSSYSSSGYGRRYASGPPASLGGPESAVA PlecHuman AAAQSTKGYYSPYSVSGSGSTTGSRTGSRTGSRAGSRRGSFDATGSGFSMTFSSSSYSSSGYGRRYASSPPASLGGPESAVA Kd of Plectin (Ex 1 -24) for actin 320 nM Kd of Plectin (R5) for vimentin (IF) 100nM Kd of Plectin (Ex 2-8) for integrin beta 4 170 nM (Ex 2-8) 25uM Kd for microtubules in case of MAP2 1 -3 uM Protein expression 5.2. Approaches for efficient protein production 5.2.1. Genetic approach x protein knowledgebase lectin N-terminal domain 1 315 ABD MTBD 848-905 SH3 1392 KRNPAHPVRGHVP rod C-terminal domain 2518 2739 3067 IFBD 1 » RDs Linker Module 4589 >ESAVA* SH3 domain Spectrin /-1 978 Actinin DM 834 Itk Tyr kinase 167 Envoplakin H 404 Actinin CE 847 Periplakin H 391 Kakapo DH 793 Kakapo AG 941 Desmoplakin H 536 HACF 8S8 Dystonin H 877 Plectin H 931 jkkhdvl JD K G E VII : ecíeey' lmgeec KHDV 3sgysy eJJ e t v dkhitc hkgdec c knd e c1 YKD D E C1 HK GD Q Ci lssih|d k S kBnD d lIsI E i h kd had P T T l d n|dli Q_kBn G E S l d n S G e v l d t S G e v KÍ1I1IEES ID1IS 5 SI ann S h ea v G f a q f s| E ad d lEDin q dkm q g p gI ed I S hd S si e tah k t s h t g p g| I S p t I S p t| l S g S hqg f v p a v gvegfvpan ghegyapss getksapaac ga e g q v p s Ink lIaB g q e g p i p |u| g sBh IvD hlJJ gn e ahv p gn e avv p s s e a a v p e k l a| e e| e V e k s Iff I P A V F pB C F V I c l l l c l l l G l I I C F l I C F T V C F l V 1037 890 £40 973 914 456 86Z 1008 S04 9Z6 944 1000 12 V. Protein expression 5.2. Approaches for efficient protein production 5.2.1. Genetic approach x protein knowledgebase Spectrin 978 Actinin DH 834 Itk Tyr kinase 1S7 Envoplakin H 404 Actinin CE 847 Periplakin H 391 Kakapo DH 793 Kakapo AG 941 Desmop1akin H £36 HACF 8E8 Dystonin II 877 Plectin H 931 PRD domain_ l Bt BedhresfqeBeetl q e eh Hs k qm l h iijqbge e^H h Hi k l k y e e e t Hl k | lhkeeq BvHEQ lkqekq BuHEQCT lkHehHdyeshkBii lkHehGdhvlkstls SH3 domain PRD 1 K G ehQd hQ 1KT 5 i ehQahQ 1ÍECHV (I Intramolecular SH3-ligand association pr ŤH K PH ) 1 Í Accessible pTyr binding site Bidemate SH2/SH3 ligand jKinvi T JEKCIVHL e C D E E Y L e G E e C T KA GD D V s e G Y s Y T eJJ E t V t D K II E T C T HECDECI C K II D E C YKD D E C QVEVT[jjHKGDQCQ| 13SIHED IESKTIIDD l d s s E i h |kd had p y t lHd l i n kJh g E s l d h s g e V l d t s g e V |kd hh E e s E d h s q e t ia h h s h e a v g p a q p s e ad d h q g ekd h gv e q d kh gh e q g p gI e t ed is ga e hd s a ghk e t a k g q e k t s kBu| t g p g gvd i s p t gh e i s p t| l s g s s s e TGHE 1 d E i 1037 e E 890 hh l z40 dB E 973 R t d 914 E T d 4s6 P P P d 8ě2 P P P d 1008 P P P H ě04 P P P H 9z6 P P P H 944 P P P H 1000 Accessible proline-rich site 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) 13 Protein expression 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 pl/Mw (average) for the protein sequence Theoretical pl/Mw: 7.78/8,726.11 mefKAIVQLKPRNPAHPVRGHVPLIAVCDYKQVEVTVHKGD QCQLVGPAQPSHWKVLSGSSSEAAVPSVCFLVPPPNQEf 14 Protein expression C-terminal domain ESAVA* 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 16 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 7.9(966 nkat) TB medium - pH 6 230 3.8 (874 nkat) TB medium - pH 7 230 CjjD (966) nkat) TB medium - pH 8 410 2.8(1,148 nkat) Additive of cellobiose (LB medium) 400 (27.0(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. 17 Protein expression 5.2. Approaches for efficient protein production 5.2.2. Expression density x functional activity The cytolinker protein: plectin Plectin is one of the main linker proteins for the cytoskeleton. ^-terminal domain Protein expression 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) Insoluble form Func. act.(45%) Func. act.(60%) Func. act. (>95%) Co-sedimentation - functional test on protein activity mix / incubate & JÉL B 4> S P S P S P kDa other proteins 45 35 25 V ř ♦ X L MTs ^centrigufal force -> supernatant | pellet protein gel analysis -I—I I R4 Kamaran Abdoulrahmari Conclusion: ♦ binds MTs, • does not . 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/ Special host system_induction method_strains required toc/IPTG or 77 I PTG Yes 77 I PTG Yes toc/IPTG No oro BAD Yes Pí/trp Yes 77 I PTG Yes P/.řeř/anhydrotetracycline No 77 I PTG Yes toc/IPTG Yes 75/1PTG Yes/TOPP 77/1PTG Yes toc/IPTG Yes Protein tag Source (website) Biotin binding domain www.promega.com His6, T7gene http://www.merckbiosciences.co.uk GST www.amershambiosciences.com His6, GFP www.invitrogen.com His6, T7 His6 www.clontech.com Chitin binding domain www.neb.com Maltose binding domain His6 www.qiagen.com Calmodulin binding www.stratagene.com peptide www.sigmaaldrich.com 20 V. Protein expression 5.2. Approaches for efficient protein production 5.2.3. Expression system x medium engineering pET32a::AHP1 pET32a::AHP5 Sea 1(4995) PVU 1(4885) Pst 1(4760) Bsa 1(4576) Earn 1105 (4357) AlwN 1(4038) 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(1S8) EcoR V(206) Nco 1(212) Msc 1(351) Rsr 11(589) Xba 1(729) SgrA 1(840) AHP - phosphotransfer protein in cytokinin signalling pathway of A. thaliana Sph 1(996) EcoN 1(1056) ApaB 1(1205) MIU 1(1521) Bel 1(1535) BstE 11(1702) Bmg 1(1730) Apa 1(1732) BssH 11(1932) Hpa 1(2027) BspLU11 1(3622) Sap 1(3506) Bst1107 1(3393) Tth 111 1(3367) BspG 1(3148) PshA 1(2366) Psp5 11(2628) pRSETB::AHP1 pRSETB::AHP5 Bands of protein of interest on SDS-PAGE Disintegration E. coli after induction Disintegration E. coli after induction Disintegration E. coli after induction Normal buffer With urea Normal buffer With urea Normal buffer With urea 100% soluble 50% soluble insoluble • Disintegrate E. coli'in native buffer and divide into two same parts. Denaturate second part of the crude extract by chaotropic compounds (urea). ►Sediment both extracts and load on 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. 22 . Protein expression 5.2. Approaches for efficient protein production 5.2.3. Expression system x medium engineering 5.2.3.1. Temperature Expression system Plasmid Temperature (°C) growth/induction Soluble form (%) Insoluble form (%) Soluble form (%) Insoluble form (%) 22°C/22°C 38% 29% 37°C/22°C 0% 100% v§2%y 18% 37°C/28°C 0% 100% 8% 92% Plasmid pET32a+ Temperature (°C) growth/induction Soluble form (%) Insoluble form (%) Soluble form (%) Insoluble form (%) 22°C/22°C /78°Ä 22% /76%\ 24% 37°C/22°C [ 67%) 33% [ 81%) 19% 37°C/28°C \61 V 39% V81%/ 19% ; Radka Fohlerová . 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 (°C) growth/induction AHP1 AHP2 AHP3 AHP4 AHP5 AHP6 37°C/28°C 8% 0% 76% 0% 37°C/22°C 73% 100% 0% 51% 22°C/22°C 71% 78% 100% 81% (£3J£) Radka Fohlerová Protein 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 6.0 Soluble fraction 35% 89% 100% Disintegrate E. coli'in native buffer and divide into two same parts. Denaturate second part of the crude extract by chaotropic compounds (urea). Sediment both extracts and load on 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. 25 Protein expression 5.2. Approaches for efficient protein production 5.2.3. Expression system x medium engineering Met 944 His 1122 pDEST17::CKI1ex1 - 371 AA, Mw = 42 kDa pDEST17::CKI1ex2 - 419 AA, Mw = 47 kDa 26 Protein expression 5.2. Approaches for efficient protein production 5.2.3. Expression system x medium engineering Growth temperature 37c 7°C, expression 28°C BL21 - 1 h after induction = OD 0.5 4 h after induction = OP 0,5 |1 h after induction = OP ? 1 4 h after induction = OD 2 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 BL21 before induction 1 h after induction = OD 0.5 13 h after induction = OD 0.5l 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 Growth and expression 25°C 1 2 3 4 5 6 ,15*^ 0 1 2 13 14 5 6 7 8 9 S: 14-66 kDa Petra Borko\#fovä 5.2. Approaches for efficient protein production 5.2.3. Expression system x medium engineering Expression E. coli BL21 (DE3) RU- TS medium: pH 6.0, pH 7.0 growth -> induction growth 25°C induction 22°C, 13.5 h Citrate b., pH 3.6, Triton X-100 Tris b., dH 7.9, Triton X-100 Lysis buffers: A. B. C. D. Tris b., pH 7.9, Triton X-100 E. Tris b., pH 7.9, CTAB F. Tris bv pH 7.9, NONIDET P-40 G. Tris bv pH 7.9, SDS H. Tris b., pH 7.9 CKhexl supernatant Protein expression 5.2. Approaches for efficient protein production 5.2.3. Expression system x medium engineering lycine pH10,6 E. coli BL21(DE3) Arctica Buffer engineering 1) pH 2) detergent Tween NP40 Western blot detected by poly- His antibodies S P Deoxycholate E- coli BL21(DE3) Arctica RP 29 Severine Jansen Protein expression 5.2. Approaches for efficient protein production 5.2.3. Expression system x medium engineering 5.2.3.3. Buffer for desintegration (pH) LTP-2 (non-specific lipid transporting protein from wheat) pH 6.0 pH 7.0 supernatant Buffer for disintegration (pH) I Glycine buffer pH 1Q.d| Phosphate buffer pH 7.2 pH6.0 pH7.0 pellet Kateřina Sikorová 30 Screening for solubility • Optimum Solubility Screen - Optimizing protein solubility and stability with salts and buffers prior to crystallization can improve crystallization results - Microbatch experiments can identify salt and buffer conditions that increase a protein's solubility Acta Cr/st. (2006) D62, 833 - 842. Izaac, C. A. Schall and T. C Mueser. Assessment of a preliminary solubility screen to improve crystallization trials: uncoupling crystal condition searcnes. Acta Cr/st (2004) D60; 1674 -1678. B. K. Collins. S. J. Tomanicek, N Lyamictieva: M. W. Kaiser and T. C. Mueser. A preliminary solubility screen used to improve crystallization trials: crystallization and preliminary X-ray structure determination of Aeropyrum pernix lap endonuclease-1 Acta Cr/st (2004) D60; 1670 -1673. J. Jancarik, R. Pufan, C. Hong, S.-H. Kim and R. Kim. Optimum solubility (OS) screening: an efficient method to optimize buffer conditions for homogeneity and crystallization of proteins. Joseph R. Luft Incomplete factorial: 950 cocktails 35 Salts and 8 Buffers Salt Dccurances j# cocktaile} KCl 31 KNÜ3 32 NhUBr 28 KH£P04 30 NH4CI 29 KSCN 29 RbCI 28 NH4N03 27 NaBr 28 NaCI 28 NK,H2PÜ4 32 28 (NH,)2HPÜ4 26 NaNQ3 30 (NK|)2S04 30 NaH2P04 23 nati 12 23 CaCb. 13 22 Li Br 32 K2HPO4 25 LiCI 3D 17 C0SO4 24 Li2SD4 27 MgCb 26 K3P04 28 MgSO,, 27 NhL,SCN 29 MnCI2 22 KC2H302 30 MnSD4 14 K Br 33 Mp(N03b 23 K2C03 34 TOTAL 950 r£ 1» 1" - ' "" '"TIL ĽKT. - I >- _ b 4 Buffer PH Occuranc*« (#cocktails} Citrate 4.2 116 Acetate 5 144 MES 6 124 MOPS 7 124 HEPES 7.5 104 Tris a 134 TAPS 9 109 CAPS 10 95 TOTAL 9SG Joseph R. Luft Data to calculate this for every proteir Statistically significant data i' I j; Incomplete factorial: 950 cocktails 35 Salts and 8 Buffers Increasing solubility Protein expression 5.2. Approaches for efficient protein production 5.2.3. Expression system x medium engineering Expression and purification of plectin's ABD (actin binding domain) in three isoforms. Protein expression 5.2. Approaches for efficient protein production 5.2.3. Expression system x medium engineering Monoclinic crystals of plectin ABD Precipitant solution: 0.1 M TRIS buffer pH 8.5 10% PEG 4000 2% dioxane Protein expression 5.2. Approaches for efficient protein production 5.2.3. Expression system x medium engineering 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 36 Protein expression 1nlv: S.M.Vorobiev, B. Strokopytov, D.G. 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). Proc.Natl.Acad.Sci. USA 100:5760-5765. 1rgi: 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. EMBO J. 23:2713-2722. 1izn: A.Yamashita, K. Maeda, Y. Maeda (2003). Crystal structure of CapZ: structural basis for actin filament barbed end capping. EMBO J. 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. 271:873-1884. Actin July 2008 PROTEIN DATA BANK 37 Die cylaskeleton is on introcellulor maze of filaments that supports and shapes Ihe tell. The most plentiful type of lilamsnt is composed of odin, shown here in blue. The tytoskelelon, however, is not a stark structure, since it must respond to the changing needs of the cell. The proteins shown here help to reshape the (ytoskcleton by assembling or disassembling nrtin filaments os necessary. A molecule of ATP, whkh is bound inside each actin molecule, is important in this process. When it is hydrofyzed to ADP, the filament becomes unstable and lolls apart. Gefsofcr breaks down actin fitements by assisting the hydrolysis of ATP and blocking the sites of interaction with other odin proteins. Two different frog-merits of gelsolin are shown in Ink and Irgi bound to odin. The protein CapZ forms a cap on the odin filaments shown in tizn, which limits assembly. The protein formin assists the assembly of actin by aligning two actin proteins in the proper irientalion which starts the process of filament growth. One domain of formin is shown bound to odin in TyM. Plectin links neighboring odin filaments into higher order structures. The actin-binding domain is shown in IshS. ThermoFluor® assay CD - spectroscopy dye interaction with hydrophobic 9 interior of protein^ native protein Temperature hydrophobic regions aggregate Joseph R. Luft ThermoFluor® assay • No data in the literature to support the prediction of crystallization conditions from Tm values • Literature reports ThermoFluor® can identify ligands that stabilize macromolecules to improve crystallization outcomes Ericsson U.B.; et af. Thermofluor-based high-throughput stability optimization of proteins for structural studies. (2006) Analytical Biochemistry 357(2) 289-298 Vedadi Ml, et at. Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination (2006) PNAS, 103(43): 15835-40 . Protein expression 5.2. Approaches for efficient protein production 5.2.5. Maximizing target protein recovery Protein expression # The knowledge applied in constructing expression vectors; •Changes in cultivation conditions: • protein induction at different temperature • media pH • various additives; Protein purification # choice of buffer for disintegrating cells (all buffers should be degassed, • The availability of a test for protein activity as a supplement to the solubility test; • The use of expression vectors without affinity fusion proteins, which one should not be afraid to do. rotein 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 rotein expression Size of the protein Formation of insoluble inclusion bodies C-terminal domain ESAVA* 4589 Mw: 8,732.3 Da H ™T-L!'-t-Ť":l'|cndorl~ in be Antibiotic selection of iransfcerants in inset: cells flashBac™ (OEiy NexiGeji Sciences) Bamd on homologous recombination at potyhedrin locus Ligasc dependent Homologous i cconibi nation in insect cells. BacVector'" IUÜ0. 2000, anon (HMD/ Novial) Based oil homologous recombination •at polyhedrin locus IJlfl1fir*vlviwTTi^rirfTi,T^i L.igasc dependent Homologous recombination in insect celts BaculoGold" (Cloentech) Bawd on homologous recombination i[ poiyhedrhi locus I.igase dependent Homologous recombination in insect ceils fiastJ ai\ homologou-'i recombination at potyhedrin Ligase dependent Homologous recombination ■:. insect cells >95% 'rotein 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 Protein expression 5.3. Expression system 5.3.4. Transient protein expression in tobacco leaves An Agrobacterium-\r\ed'\a\ed 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 scFvx DHZR in Tobacco 17 «8 «9 «10 11 12 BIG 18 62,7 66 51,0 huh 8 9 10 11 12 16 47 Before expression >Host strain (e. coli, Yeast, mammalian cell, plant) >Expression vector (piasmid) >Gene construct: ❖domain and secondary structure prediction ❖codon usage ❖stability ❖toxicity >Buffer options for disintegration