Membrane Protein. Expression Purification Reconstitution Sample Preparation

Transcription

1 Membrane Protein Expression Purification Reconstitution Sample Preparation Tim Cross, Florida State University - if interested in help with any of the above contact us and arrange to spend a few days in my lab with people who have lots of first hand experience (i.e. not myself). Preserving your sample Tim Cross, National High Magnetic Field Lab - if interested in this technology - please contact myself of Bill Brey at the NHMFL. Sensitivity, Sensitivity, Sensitivity,

2 Membrane Protein Expression and Purification Flow Chart Data above for Overexpression can be substantially improved

3 Engineered Vector N-terminal His Tag, Fusion partner insertion site, TEV cleavage site, LIC site, Target sequence

4 Expression as a Function of Vector

5 To Illustrate Expression, Purification, and Reconstitution: Rv1861 from M. tuberculosis 11.4 kda monomer amino acid residues 3 putative transmembrane helices therefore little extramembranous protein Forms a well defined oligomeric state on PFO gels Has GTP binding ability and potentially is a Molybdenum transporter This construct has an N-terminal His6 tag and ampicillin resistance.

6 Membrane Protein Expression: I 50 ml culture of E. coli in LB media initiated with 5 transformed cells from a - 80 C stock (don t let the stock thaw). Add ampicillin Allow the culture to grow for approximately 16 hours at 37 C Document - sample for SDS-PAGE, OD600 Transfer 20 ml to each of two 1L of LB media, add ampicillin OD Grow at 37 C until an OD600 of 1.5 Document - sample for SDS-PAGE, OD600 Harvest cells from LB media at 6000 g for 5 min, Decant supernantant and invert centrifuge tubes on a paper towel for 2-3 min M9 media prepared with glucose (do not autoclave the glucose, filter the solution) 30 min Prior to transfer to M9 media warm the media to 37 C Time (hours)

7 Membrane Protein Expression: II Resuspend cells with 10 ml of M9 media. Transfer to M9 flasks. Add ampicillin Grow cells at 37 C for min Document - sample for SDS-PAGE, OD600 Add 0.4 mm IPTG and continue to grow at 37 C Time (hours) (for some proteins need to grow cells to high OD prior to inducing) (for some proteins growth at a lower temperature may induce proteins to be expressed in the membrane and may result in a higher yield) Harvest cells when they reach maximum OD600 (4-6) which takes 3-5 hours - Document - sample for SDS- PAGE, OD600 Collect cells as before. Scrape cells into freezer box. Cells can be kept at -80 C for up to a month Lane 1: Ladder; Lane 2: Overnight Culture; Lane 3&4: LB Cultures at OD600 at 1.5; Lane 5 &6: M9 Before Induction; Lane 7&8: Harvest OD IPTG

8 Membrane Protein Solubilization: I Lysis can be done French Press or chemical means Calculate an appropriate Lysis Volume: LV(mL)= Culture Vol. {OD600/100} Thaw cells by adding buffer (20 mm TRIS ph mM NaCl) to the cells to a 80% of the total LV Homogenize the solution by passing the suspension through an 18 gauge needle. Add 10% LV in the form of a Lysis Buffer (20 mm TRIS, ph 8.0, 1% Triton, 8M Urea Add 2 µl/l of culture volume benzonase. Add lysozyme to 0.25 mg/ml of LV Incubate suspension for 30 min at RT Document - sample for SDS-PAGE Add Empigen 3% from 35% stock (approximately 10% of LV) Other proteins may be better solubilized with different detergents - a detergent screen at this point may be useful - the LV can be divided into many aliquots

9 Membrane Protein Purification: I Mix well on stir plate and incubate at 4 C for at least 12 hours document with sample for SDS-PAGE Centrifuge at 12,000 g for 20 min, transfer supernatant document supernatant with sample for SDS-PAGE Equilibrate AKTA column with buffer (20 mm TRIS, ph 8, 500 mm NaCl, 0.7% empigen) Load supernatant and wash with equilibration buffer until the flow-through has a baseline OD280. Note the volume of flow-through document with sample for SDS-PAGE Wash column with 40mM imidazole added to the equilibration buffer until OD280 returns to baseline document with sample for SDS-PAGE Exchange empigen for SDS. Wash with two column volumes using 0.2% empigen in place of the 0.7% empigen in 20 mm TRIS, ph 8. Wash with two column volumes of 20mM TRIS, ph 8, 0.25% SDS Document with sample for SDS-PAGE

10 Membrane Protein Purification: II Elute with 20 mm TRIS, ph 8, 0.5% SDS, 500 mm imidazole. Capture the OD280 peak in a single fraction The amount of imidazole can vary from protein to protein Document with sample for SDS-PAGE Lane 1: Ladder Lane 2: Lysate Lane 3: Lysate + Empigen Lane 4: Supernatant Lane 5: Flow-through Lane 6: Wash Lane 7 Elution»» Note that the intensities are comparable

11 Membrane Protein Reconstitution: I Transfer 8 mg Rv1861 in 0.5% SDS, 500 mm imidazole, 20 mm TRIS, ph 8 to a tube Document with sample for SDS-PAGE Add SDS to make the protein solution 2.3% (w/v) SDS. This concentration is critical as discussed on the next slide Calculate the amount of lipid necessary for a 1:200 molar ratio of protein to lipid. Here we use 4:1 molar ratio of DMPC and DMPG to provide the bilayers with significant negative charge. Vortex 95 mgs of lipid and 3 ml nanopure water. Bath sonicate at 35 C (above the gel to liquid crystalline phase transition) for min. The solution should be translucent, but not transparent. Add 20% SDS solution to the lipid preparation to a final concentration of 2.3% (w/v). Mix the solution gently. A clear but very viscous solution results. Mix the Lipid and Protein solutions on a rocker at room temperature for 20 min. The solution should become very fluid Document with sample for SDS-PAGE

12 Lipid Detergent Solution: Lipid/Detergent solutions can be considered to have three stages - Detergent monomers are incorporated into the liposomes (extensive light scattering) - When monomers saturate the liposomes lipids start to become integrated into micelles (solid black arrows). Viscosity increases to this point. - When all of the lipid is integrated into micelles the light scattering plummets (white arrows) a. Triton X-100 b. Octyl Glucoside c. Sodium Cholate»» In the process of mixing the Lipid and Protein solutions the lipids become micellarized a. c. b. Paternostre et al., 1988 Biochem. 27:

13 Membrane Protein Reconstitution: II Incubate the protein/lipid/sds mixture at 37 C for hours without rocking or shaking Centrifuge g for 20 min to eliminate any precipitate. Decant supernatant immediately Document with sample for SDS-PAGE Load sample into a 3.5 kda dialysis bag. Dialyze at room temperature against 2L of 20 mm TRIS ph 8. Change buffer twice/day and dialyze for 7-14 days. Sample should become translucent Amberlite or biobeads may enhance the rate of this step. After dialysis note the volume of the protein solution. Document with sample for SDS-PAGE Centrifuge g for 20 min - there should be no precipitate. Decant supernatant immediately Centrifuge g for 2 hours at 4 C to pellet the proteoliposomes. Decant. Document that the supernatant has no protein with a sample for SDS-PAGE

14 Membrane Protein Reconstitution: III Resuspend proteoliposomes in 20 mm TRIS ph 8 such that 40 µl of solution contains 2.5 mg lipid (the amount used per glass slide) Centrifuge g for 20 min to eliminate any precipitate. If there is a pellet check via bath sonication if it goes into solution or if it is a precipitate. Do not use proteoliposome samples containing a precipitate. Deposit 40 µl of proteoliposomes per slide spreading the sample out with a pipet tip. Allow sample to dry overnight and then rehydrate in a humidity chamber at 98% relative humidity after stacking the slides. The intensity of the Rv1861 band remains constant except for the last lane that indicates some protein was lost due to precipitation and consequently the molar ratio of protein to lipid in greater than 1:200.

15 Oriented Sample Preparation on Glass Substrate: General Strategy The basic idea for oriented sample preparation is to induce alignment of bilayers with respect to the glass surface. assembly of lipid molecules into a planar bilayer on the surface annealing liposome preparations onto the surface. Bilayer assembly is typically not viable for proteins as opposed to peptides, so reconstitution of full length protein into liposomes is necessary step. Hydrophobic peptides are usually soluble and stable in organic solvents and relatively easily aligned in lipid bilayers by rehydrating a peptide/lipid film to induce bilayer assembly - I will not discuss this further. Once fully hydrated the viscosity is such that alignment of the samples can be enhanced by placing the sample in the magnetic field. It is assumed that this effect is small when very high ratios of lipid to protein are required.

16 New Sample Sealing Mechanism

17 MP Aligned Sample Preparation: I Glass slides ( ours are 5.7 x 11.5 mm) are washed with chloroform, methanol and water then dried in the oven. Measure the weight of dry stacked slides, place the sample cell in 98% humidity chamber ( saturated solution of K 2 SO 4 ) at 37 C. Monitor the weight gain until it stabilizes, seal the sample cell with a glass slide and wax. Usually we hydrate our samples for 5-7 days for DMPC:DMPG mixtures. Well hydrated samples usually are somewhat transparent. Water droplets forming on the sample cell wall is an indication that there is severe sample heating taking place: The recycle delay and cooling air are not particularly effective in controlling this problem. 31 P is only partially effective for quality control - signals from the protein are needed for assessing the quality of alignment.

18 The Precision of Time Averaged Orientational Restraints»» 2 H spectra of d 5 Trp labeled gramicidin A in uniformly aligned lipid bilayer preparations.»» The linewidths are consistent with an orientational dispersion of just ±0.3 - consequently very precise indole orientations. This is similar to the dispersion in protein crystals

19 MP Aligned Sample Preparation: II Factors affecting choice of lipids: - Lipid chain length : Hydrophobic thickness, tilt angle. - Phase transition temperature: Protein exposure to high temperature may not be desirable, but experiments need to be performed at temperature higher than T m, adds to heat generated by high RF field. - Ester linkages in lipids are prone to hydrolysis, for longer stability of oriented samples ether linked lipids can be used. - Inter bilayer separation is dependent on bilayer charge and can be modulated by using ionic lipids. This inter bilayer separation plays an important role if the protein has a large domains outside the membrane. PG appears to be helpful in a number of membrane protein preparations.

20 Bilayers as Models of Native Membranes Bilayers provide a heterogeneous environment typical of the native membrane. Bilayers provide a dielectric gradient from the lipid interfacial region to the low dielectric at the center of the bilayer Bilayers provide a water concentration gradient typical of native membranes Bilayers provide a range of fluidity typical of native membranes, because sample temperatures will approximate native conditions. Bilayers provide a hydrophobic dimension that matches that of the protein

21 Bilayers as Models of Native Membranes, but Bilayers will not have the same complex mixture of 100 or more different lipids in native membranes. Bilayers will not have an asymmetric distribution of lipids between the bilayer leaflets Bilayers do not have the complex glycosylation that native membranes have Bilayers may not display the same gradient of lateral pressure Bilayers may not display the same position on the complex phase diagram consistent with a given level of curvature frustration Bilayers will not have the native-like chemical, electrical, and mechanical potentials across the bilayer that is typical of native membranes. Therefore, we need to be careful about how well we say that we model the membrane environment Nevertheless, these bilayer preparations represent an environment that is much more native-like than any of those utilized by the other structural methods today.

22 FSU Chem & Biochem, Inst. of Mol. Biophys. Mukesh Sharma Milton Truong Dylan Murray Nabanita Das Thach Can Anna Kozlova Huajun Qin Dr. Sorin Luca Dr. Da Qun Ni Dr. Frantz Jean-Francois FSU Mathematics Prof. Jack Quine Prof. Richard Bertram FSU Physics Myunggi Yi Prof. Huan-Xiang Zhou NHMFL Dr. Riqiang Fu Dr. William Brey Peter Gor kov Brigham Young Univ. Prof. David Busath Hamilton College Prof. Myriam Cotten The NHMFL: A National User Facility

23 RF Heating of Lipid Bilayer Preparations is Particularly Significant Dielectric Losses Inductive Losses Heating is primarily from the 1 H Channel At modest ion concentrations the heating is independent of ion concentration The heating is highly dependent on hydration Dielectric Losses dominate Inductive Losses Li et al., 2006 JMR 180:51; Gor kov et al., 2007 JMR 185:77

24 Sample heating in solenoids Temperature rise in mechanically aligned preparation after single 1 H decoupling pulse: 50 khz, 10 ms long T T ( C) flat coil sample»» measured using the indicator TmDOTP B 0 field (MHz) S. McNeil et al., Magn. Reson.. Chem. (2007), in press

25 Reduction of 1 H decoupler heating typical buffer range Different buffer concentration Bicelle probes, 600 MHz Solenoid, 5t Sample heat absorption (mw/khz 2 ) Low-E, 8t NaCl concentration (mm) Data by C. Qian