Fig 1.
LoFT (lysozyme treatment, osmotic shock, and freeze-thawing) cell extraction protocol.
(A) A typical protocol to prepare the LoFT cell extract. (B) Coomassie staining after SDS-PAGE, showing the expression of GFP and FtsZ (a bacterial tubulin homolog) using the LoFT cell extraction protocol. pOR2OR1-sfGFP (gfp under control of the OR2-OR1 promoter), pET29-sfGFP (gfp under control of the T7 promoter), or pET29-ftsZ (ftsZ under control of the T7 promoter) plasmid was mixed with LoFT cell extract and reaction mixtures and incubated for 14 h at 29°C. Expression of FtsZ was confirmed using a FluorTect GreenLys in vitro translation labeling system (Promega, Fitchburg, WI).
Fig 2.
Conditions of freezing and thawing for LoFT cell extraction.
(A) Effects of freezing temperature on protein concentration (protein conc.) after freeze-thawing. Samples were frozen in a deep freezer (-80°C) for 1 h, immersed in liquid nitrogen (Liq. N2) for 15 min, or not frozen. (B) Protein concentration (gray bars) in the LoFT cell extracts and volume of supernatant (Sup.) after centrifugation (circles) compared to the number of freeze-thaw cycles. Collectable supernatant volumes were derived from the viscosity of cell debris after freeze-thaw. Because the standard deviations of the collectable solution (n = 3) were smaller (on the scale shown) than the circle sizes, error bars are not indicated. All samples in (A) and (B) were thawed by incubation in ice water, and error bars indicate standard deviation (n = 3).
Fig 3.
Effect of centrifugation force on LoFT cell extract.
Protein concentration (gray bars) and levels of sfGFP expression, from CFPS using LoFT cell extracts and 3 nM pOR2OR1-sfGFP (circles), were plotted against forces of centrifugation (12,000 × g for 2.5 h, 16,000 × g for 94 min, and 25,000 × g for 1 h) after freeze-thawing. Error bars indicate standard deviations (n = 3). Expression levels of sfGFP were normalized to the average value of the cell extracts obtained after centrifugation at 25,000 × g for 1 h (vertical axis). GFP exp. means “GFP expression”.
Fig 4.
Essential chemicals for CFPS using the LoFT cell extract.
The importance of each chemical for GFP expression was evaluated by systematic omission from the reaction mixtures. AA indicates the mixture of 20 amino acids. Template DNA used was 10 nM pOR2OR1-sfGFP. CFPS was performed at 29°C for 14 h. In the condition indicated by “all,” none of the chemicals were omitted. Error bars indicate standard deviations (n = 4).
Fig 5.
CFPS using a linear DNA template and LoFT cell extract.
sfGFP was produced using LoFT cell extract and 5 nM linear DNA (PCR product) for 1 h at 29°C, in the absence (−) or presence (+) of 4 μM GamS. OR and T7 indicate PCR products from the OR2OR1-sfGFP (from the pOR2OR1-sfGFP plasmid) and T7-sfGFP (from the pET29-sfGFP plasmid), respectively. Prom. means “Promoter”. (A) Expression levels of GFP estimated by total fluorescence levels. Error bars indicate standard deviations (n = 3). (B) GFP fluorescence detected using SDS-PAGE without boiling. Only bands corresponding to GFP fluorescence are shown.
Fig 6.
Essential chemicals for CFPS using LoFT cell extract after buffer exchange.
The importance of each chemical for GFP expression was evaluated by systematic omission from the reaction mixtures. AA indicates the mixture of 20 amino acids. pOR2OR1-sfGFP (10 nM) was used as the DNA template. CFPS was performed at 29°C for 14 h. “all” indicates no chemicals were omitted. Expression levels of sfGFP were normalized to the average value of “all.” Error bars indicate standard deviation (n = 4). Expression of sfGFP without GluK and Mg is a result of the exchange buffer (S30 buffer) containing these chemicals.
Table 1.
A comparison of cell-free extracts prepared by physical disruption and by the LoFT method.