Strategies for Protein Expression and Analysis

Transcription

1 Strategies for Protein Expression and Analysis Ute Boronowsky, Ph.D. Senior Global Product Manager Protein Technologies QIAGEN - 1 -

2 QIAGENs Solutions for Proteins Along the recombinant workflow Template generation Expression Purification Assay & Crystallization - 2 -

3 QIAGENs Solutions for Proteins Along the recombinant workflow Template generation Expression Purification Assay & Crystallization - 3 -

4 Rational gene design and de novo synthesis Comparative Studies Rational gene design (multi-parameter optimization of the coding DNA sequence codon usage, mrna sec. structures, G/C content...) vs. Cloning of wild-type cdna Is it really better? 2 multi-gene studies (100 human proteins each, optimized and wt genes each) 70 % of optimized genes expressed better than wt cdna (E. coli) 90 % of optimized genes expressed better than wt (HEK293) Maertens et al. (2010). Protein Science Fath et al. (2010). Manuscript subm. PepTalk

5 Rational gene design and de novo synthesis QIAgenes Expression Constructs The QIAgene concept: Gene & vector design Synthetic, sequence-optimized human ORFs cloned into a standardized vector for enhanced expression yields in E. coli or insect & mammalian cells E. coli in vivo E. coli cell-free Mammalian cells in vivo Insect cells in vivo Euk. cell-free systems (Insect, Reticulocyte lysates) Any human gene available as QIAgene (E. coli & Insect/Mammalia: 2x 35,000 constructs) a cooperation project with Protein Science Forum Nov 9 th

6 PlasmidPlus for template purification PlasmidPlus Kits High yields: 250 µg (Midi) 1 mg (Maxi) Fast: Only 20 min to pure plasmid! Low endotoxin levels for sensitive cell transfection NEW: PlasmidPlus 96 Kit Same high DNA quality >20 µg DNA (Mini) Automated or manual procedure - 6 -

7 QIAGENs Solutions for Proteins Along the recombinant workflow Template generation Expression Purification Assay & Crystallization - 7 -

8 Protein Expression Systems Which one? E. coli cell-free (fast, scalable to 10 mg, co-translational labeling/nmr) in vivo (BL21, C41/43; cheap) Insect cells (Sf9, Sf21) cell-free (fast, membrane-incorporated, functional analyses) in vivo (transient plasmid transfection, Baculovirus transfection) Yeast (Saccharomyces, Pichia) Mammalian cells (HEK293, CHO) (good functionality, expensive, low yields) New systems (Lactococcus lactis, Tetrahymena...) PepTalk

9 Cell-free Expression of Proteins in a Day Template Generation Protein Synthesis EasyXpress cell-free extract on standard thermoshaker Analyze Protein - 9 -

10 Gene Optimization for Membrane Proteins System Protein wt optimized Factor TLR Toll-Like Receptor 10 (94 kda, 1 TM) E.coli in vivo OPRM1 1.5 Opiod Receptor (45 kda, 7 TM/GPCR) TRPV4 >50 Transient Receptor Potential Cation Channel (98 kda) Insect cell-free KCNJ1 >50 Potassium Channel SERT >50 Serotonin Transporter (70 kda) HEK293 in vivo TLR10 >50 TLR10 Aqp5 9.0 Aquaporin 5 (28 kda) Data from systematic study with 100 genes: With optimized genes... Membrane Proteins can be successfully expressed in all tested expression systems Maertens et al. (2010). Protein Science Fath et al. (2010). Manuscript subm

11 Why cell-free expression with EasyXpress? Fast No need for specialized equipment Standardized for reproducible results Suitable for membrane proteins Suitable for proteins with disulfide bridges (e.g. antibody fragments) Open system: Ideal for co-translational labeling with stable isotopes for NMR addition of detergents, cofactors... possible

12 QIAGENs Solutions for Proteins Along the recombinant workflow Template generation Expression Purification Assay & Crystallization

13 Challenges in Recombinant Protein Purification Purity & yield Solubility of protein Different proteins behave differently Robust process Scalable process Several affinity purification technologies to address these challenges

14 3 Major Purification Technologies from QIAGEN Ni-NTA Superflow & Antibodies Proven, robust technology NEW: Glutathione Superflow & Antibody Purification of GST-tagged proteins GST pull-down assays Enhances protein solubility FPLC compatible NEW: Strep-Tactin Superflow Plus & Antibody Purification of Strep-tagged proteins Well suited for eukaryotic expression systems 3-fold increased binding capacity Bulk resin

15 6xHis-tagged Protein Purification Native conditions Tris or phosphate buffer, ph mm NaCl mm imidazole Cell lysis Denaturing conditions Phosphate buffer, ph 8 8 M urea or 6 M GuHCl (imidazole optional) min (batch or column format) Ni-NTA resin Bind Wash min (batch or column format) mm imidazole Elute ph mm imidazole Highly pure 6xHis-tagged protein ph 5.9 or ph

16 Efficient one-step purification with Ni-NTA Highly pure proteins after purification with Ni-NTA Superflow The indicated proteins (arrowed) were expressed in E.coli using QIAgenes Expression Constructs. Proteins were purified in parallel using QIAGENs Ni-NTA Superflow or a nickel resin form supplier G. CL: Cleared lysates, Each set of three lanes shows form left to right: flow-through, wash and elution fractions

17 High protein binding capacity of Ni-NTA resins High yields of up to 50 mg/ml protein Binding of various his-tagged proteins of Ni-NTA: binding was performed in bath procedures and proteins quantified using the Bradford method

18 Reagents compatible with Ni-NTA resins Ni-NTA is the only resin that is compatible to 10 mm DTT

19 NEW Ni-NTA Membrane Protein Kit For solubilization and purification of membrane proteins Complete kit Standardized procedure Easy-to-follow protocols for E.coli or Insect culture or cell-free extracts Box 1 Resin & Buffers Box 2 7 detergent aliquots

20 Purification of Membrane Proteins with Ni-NTA In vivo and Cell-Free Expression - Examples In vivo expression of optimized human Caveolin-1 (Caveolar scaffolding protein 1, 1 TM, 21 kda) Cell-free expression of optimized human MAL (Myelin and Lymphocyte protein, 4 TM, 16.7 kda) L R FT W E S I R FT W E DDM Triton X-100 Ni-NTA Superflow purification in the presence of detergent: L: Total lysate; R: Detergent-resolubilized fraction; FT: IMAC flow-through fraction; W: IMAC wash fraction; E: IMAC elution fractions; S: soluble protein; I: insoluble protein Kubicek, Spriestersbach, Maertens PepTalk

21 Scalable Purification with Ni-NTA From screening to bulk production

22 Let your instrument do your sample prep for you

23 Summary: Features & Benefits of Ni-NTA Technology 6xHis tag is very small usually no need to remove it Purification under native and denaturing conditions High yields of pure proteins: up to 50 mg/ml resin Very robust technology Specific Kit for purification of 6xHis-tagged membrane proteins

24 3 Major Purification Technologies from QIAGEN Ni-NTA Superflow & Antibodies Proven, robust technology NEW: Glutathione Superflow & Antibody Purification of GST-tagged proteins GST pull-down assays GST-tag antibody FPLC compatible NEW: Strep-Tactin Superflow Plus & Antibody Purification of Strep-tagged proteins Well suited for eukaryotic expression systems 3-fold increased binding capacity Bulk resin

25 Purification of GST-tagged Proteins Starting material: cleared lysate from E.coli, Insect or mammalian cell-culture or cell-free extracts Bind Cleared lysate to equilibrated resin Wash Elute with reduced glutathione

26 One-step purification with Glutathione Superflow

27 Features & Benefits of Glutathione Superflow The GST-tag (26 kda) increases protein solubility Mild elution conditions maintain protein function Easy to use Suitable for pull-down assays GST-Tag/Glutathione Superflow: the technology of choice for insoluble proteins

28 3 Major Purification Technologies from QIAGEN Ni-NTA Superflow & Antibodies Proven, robust technology NEW: Glutathione Superflow & Antibody Purification of GST-tagged proteins GST pull-down assays GST-tag antibody FPLC compatible NEW: Strep-Tactin Superflow Plus & Antibody Purification of Strep-tagged proteins Well suited for eukaryotic expression systems 3-fold increased binding capacity Bulk resin

29 StrepTactin Plus Purification Resin Strep tag II: 8 amino acid peptide W-S-H-P-Q-F-E-K interacts with Strep-Tactin: engineered Streptavidin Mild elution with biotin analogue Purification of functionally active proteins Suitable for purification from mammalian cells Plus: Now with 3fold higher binding capacity

30 Purification of Strep-tagged Proteins Starting material: cleared lysate from E.coli, Insect or mammalian cell-culture or cell-free extracts Bind cleared lysate (E.coli / insect / mammalian cell culture or cell-free extract) Wash Elute with desthiobiotin

31 One-step Purification on StrepTactin resin Strep-GFP Strep-tagged GFP was expressed in E.coli culture and applied to Strep-Tactin Superflow Plus. Total protein yield was 3.1 mg. C: Cleared lysate; E: Elution fractions; F: Flow-through; M: Markers; W: Wash From TI /

32 Overview Affinity Purification Technologies NEW NEW

33 Superflow Cartridges for Purification by FPLC Prepacked columns for Liquid Chromatography Available for all purification resins 1 ml and 5 ml size Robust construction allowing high flow rates Compatible to commonly used FPLC Systems (ÄKTA, BioLogic)

34 Highly Specific and Sensitive Antibodies

35 QIAGENs Solutions for Proteins Along the recombinant workflow Template generation Expression Purification Assay & Crystallization

36 Protein stability and Ligand binding measurements: Differential Scanning Fluorimetry (DSF) on a real-time PCR cycler Applications: Identification of protein stabilizing conditions For storage For crystallization Ligand binding, K D measurement Mutant protein analysis -> 2 application notes on QIAGEN website Features: Small sample size: 5-10 µg protein/well Up to 100 samples in parallel High-resolution cycler with HRM channel standard SYPRO Orange dye

37 Differential Scanning Fluorimetry (DSF) principle

38 Rotor-GeneQ: Best suited instrument for protein melting applications Benefits of the Rotor-GeneQ: Outstanding thermal and optical performance due to rotary format User-friendly software Low maintenance and maximum convenience due to robust design The rotary design delivers: Well-to-well variation below ± 0.01 C (20 times less than block cyclers) Fast ramping and negligible equilibration times for short run times The High Resolution Melting (HRM) option for the Rotor-Gene Q includes: A specially tuned high-intensity optical HRM channel Temperature accuracy: ±0.25 C High data acquisition rates

39 Stability measurements of a range of proteins Raw data showing increase of fluorescence of SYPRO orange Negative first derivatives are used to estimate the melting points Loss of thermostable DNA polymerase activity

40 QIAGENs Solutions for Proteins Along the recombinant workflow Template generation Expression Purification Assay & Crystallization

41 Crystallization Workflow: 1. Solutions Pre-Screen Assay Initial screen Fine screen Optimization & Upscale EasyXtal Pre- Screen Assay (24-well prefilled plate) JCSG Core I-IV PACT Classics, Class. II, Class. Lite, Cryo Anions + Cations PEG I + II AmSO4 MPD ComPAS Stock Solutions (50 / 200 ml) or: Refill Hits (50 ml) + Pre-Screen Assay phclear I+II in 96 x 10 ml Tubes or 96 x 1,5 ml Deepwell Blocks OptiSalt Suite (0.5 ml DWB) Stock Tubes Deepwell Block (DWB) RefillHit

42 The world s largest offer of crystallization reagents: 24 crystallization screens with >2000 conditions!

43 Production reports: High Quality Control Standard Available online for every crystallization solution Detailed protocols Detailed information on supplier, purity grade, cat.# etc. Concentration and ph of buffer stock solution, Counterion Exact composition including final ph

44 Crystallization Workflow: 2. Plastics Evolution µplate EasyXtal Tool # of wells Screening? Yes Limited LLOptimization? Yes Yes Upscale? Yes, max Optimization 1+1 µl drop Upscale Yes Setup? Manual or automated Manual Readout? Manual or automated Manual or automated

45 Automatable Membrane Protein Crystallization NeXtal Evolution µplate + Mono-olein = NeXtal CubicPhase µplate READY TO USE! for 450 nl protein FULLY AUTOMATABLE solution per well Cubic Phase Monoolein for 100 nl protein solution per well (MRC 2-well / IntelliPlate 3-well ) Monoolein / Cholesterol mixes e.g. for GPCR crystallization

46 Membrane Protein Crystallization Simple Vapor Diffusion Setup

47 Crystals from Bacteriorhodopsin Diffract to 1.1 Ǻ 1.1 Å 1.0 Å High-resolution data measured in Grenoble ESRF Bacteriorhodopsin crystallization conditions: Na/KPO 4 /H 2 O Screen: M Na/KPO 4 ; ph 4-10 Kubicek, Labahn

48 Crystallization of Sensory Rhodopsin II a New 7 Transmembrane Protein Structure 1 well of a SRII LCP crystallization experiment (NeXtal CubicPhase) 100 µm SRII crystals 2.7 A resolution (ESRF, Grenoble) Reaction Setup: 0,45 µl protein 0,45 µl buffer 70 µl precipitation reservoir Structure determination Kubicek, Labahn, Schlesinger

49 QIAGEN s Solutions for Easy Access to Proteins Along the recombinant workflow Template generation Expression Purification Assay THANK YOU!