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Cell-free expression of integral membrane Cell-free expression of integral membrane proteinsproteinsCell-free expression of integral membrane Cell-free expression of integral membrane proteinsproteins
Christian Klammt, Innokentiy Christian Klammt, Innokentiy Maslennikov, Maslennikov, Witek Kwiatkowski and Senyon ChoeWitek Kwiatkowski and Senyon ChoeThe Salk Institute, Structural Biology Laboratory, La Jolla, The Salk Institute, Structural Biology Laboratory, La Jolla, CACA
Protocols for set-up of an E. coli based CF expression system– S30 cell extract preparation– T7-RNA polymerase preparation– List of reaction components– Stock solutions of CF components– Pipetting protocol– Reaction compartments for dialysis mode
CF expression of integral membrane proteins (IMPs)– Possible modes for CF IMP expression
– Inoculate 5 liters of TB media at a ratio of 1:100 with fresh overnight culture of E. coli strain (A19, BL21, D10) in a fermentor and start rapid cooling when cells reach log-phase (OD 600 = 5-6). All following steps on ice!
Cell wash:– Harvest cells by centrifugation (7,000 x g, 4°C, 10 min) and resuspend in Buffer A
(10 mM Tris-Acetat, pH 8.2, 14 mM Mg(oAc)2, 0.6 mM KCl, 6 mM BME) using a glass rod.
– Centrifuge cells (8,000 x g, 4°C, 30 min) and repeat washing, [STORE ON ICE OVER NIGHT], wash 3rd time.
Cell lysis (day 2):– Weight cells and resuspend in 110% w/v buffer B (10 mM Tris-Acetat, pH 8.2, 14 mM Mg(oAc) 2,
0.6 mM KCl, 1mM DTT, 0.1 mM PMSF). Disrupt cells by French press (20,000 psi) or cell disrupter at 4°C.
Isolation of cell extract:– Centrifuge disrupted cells (30,000 x g, 4°C, 30 min), keep first 2/3 of supernatant and centrifuge again.
keep 2/3 of second supernatant.
Run-off procedure:– Supplement extract with 400 mM NaCl (5 M stock) and incubate for 45 min at 42°C.
Dialysis:– Dialyse against 2x 40 volumes over night with first buffer exchange after 2 h into S30 buffer (10 mM
Tris-Acetat, pH 8.2, 14 mM Mg(oAc)2, 0.6 mM KoAc, 0.5 mM DTT at 4°C (25 kDa MWCO membrane).
Store extract (day 3)– Clear extract by centrifugation (30,000 x g, 4°C, 30 min), aliquot and freeze in liquid N 2, store at -80°C.
– Inoculate 4 liters of LB in Erlenmeyer culture flasks at a ratio of 1:100 with fresh over night culture of BL21 Star x pAR1219. Incubate at 37°C with vigorous shaking until OD600 of 0.6-0.8. Induce T7RNAP with 1 mM IPTG and incubate at 37°C with vigorous shaking for 5 h.
– Harvest cells by centrifugation (4,500 x g, 4°C, 15 min) and resuspend cell pellet in 120 ml T7 buffer (30 mM Tris, pH 8, 10 mM EDTA, 50 mM NaCl, 5% Glycerol, 10 mM BME).
Cell lysis:– Disrupt cells with French press (20,000 psi) or cell disrupter. Adjust the supernatant to final
concentration of 2% streptomycin sulfate by gentle stirring and drop wise addition of 10% stock.
– Remove precipitated DNA from supernatant by centrifugation (30,000 x g, 4°C, 30 min).
Purification:– Purify T7RNAP by anion exchange chromatography. Equilibrate Q-Sepharose column (1.6 x
10 cm) with T7 buffer, load supernatant with 1ml/min flow rate.– Elute T7RNAP in gradient from 50 mM to 500 mM NaCl in 15 column volumes at a 3 ml/min
flow rate. T7RNAP starts to elute at ~150 mM NaCl.
Dialysis:– T7RNAP fractions are pooled and dialyzed against 2x 100 volumes of dialysis buffer
(10 mM Tris, pH 8, 1 mM EDTA, 10 mM NaCl, 10% glycerol, 1 mM DTT) over night.
Activity test (day 2):– T7RNAP activity is tested by in vitro transcription and units are determined by comparison
with a commercial control.
Store T7RNAP:– Store T7RNAP in aliquots in dialysis buffer adjusted to 50% Glycerol at -20°C.
L-CF: direct translation into bicells, lipids, NLPs, preformed liposomes
(P-CF) (D-CF) (L-CF)
3 possible modes for CF IMP expression
Klammt et. al 2007: Cell-free expression of integral membrane proteins for structural studies. In: Cell-free expression techniques, A. Spirin and J. Swartz (eds.), Wiley-VCH, Weinheim, Chapter 8, pp.141-164.
– Verify efficiency of CF system by expression controls (e.g., with GFP)
– Ensure high quality of template DNA– Modify DNA template design (e.g., addition of expression tags)– Analyze mRNA secondary structures to ensure efficient initiation
and elongation of translation– Ensure compatibility of extra compounds supplied with CF system
Low IMP solubilization in D-CF– Evaluate a series of detergent types– Optimize the final detergent concentration– Provide detergent mixtures or combinations of detergents/lipids– Decrease expression temperature
Production of IMP fragments– Stabilize IMP by protease inhibitors during CF expression– Increase tRNA concentration– Use S30 extracts from protease-negative strains