Min protein in vitro protein expression kinetics
Godino E, López JN, Foschepoth D, Cleij C, Doerr A, Castellà CF, Danelon C. De novo synthesized Min proteins drive oscillatory liposome deformation and regulate FtsA-FtsZ cytoskeletal patterns. Nature Communications. 2019 Oct 31;10(1):1-2.
- Organism: Escherichia coli
- Instrument: 6460 Triple Quadrupole LC/MS
Min system, CFPS, in vitro gene expression, MRM, SRM,
The Min biochemical network regulates bacterial cell division and is a prototypical example of self-organizing molecular systems. Cell-free assays relying on purified proteins have shown that MinE and MinD self-organize into surface waves on a planar membrane and into various oscillatory patterns in closed compartments. In the context of developing a synthetic cell from elementary biological modules, harnessing Min oscillations might allow us to drive higher-order cellular functions. However, to convey hereditary information in a synthetic cell, the Min system must be encoded in a genomic DNA that can be copied, transcribed and translated. Here, the MinD and MinE proteins are de novo synthesized from their genes inside liposomes. Dynamic protein patterns and liposome shape deformation accompanying Min protein recruitment to the membrane are observed. By enabling genetic control over Min protein self-organization and membrane remodeling, our methodology offers unique opportunities towards directed evolution of bacterial division processes in vitro.
A targeted proteomics approach was used following established in-house protocols. A sample of a PUREfrex 2.0 reaction (GeneFrontier, Japan) of 5 µl was taken and incubated at 90°C for 10 minutes in 22.65 µl of 27.56 mM Tris-HCl pH 7.6, 4.5 mM Dithiotreitol (DTT) and 1.1 mM CaCl2. To make sure some of the expressed protein was in the linear regime of the standard used another 2.5 µl and 1.25 µl of expression was used and diluted to a final volume of 5 µl giving a 2x and 4x dilution of the expression. Then 15.52 mM final concentration iodoacetamide is added and the solution is incubated for 30 minutes in the dark. The iodoacetamide reaction is quenched by additional addition of 4.2 mM final concentration of DTT. Finally 0.625 µg of Trypsin is added and the solution is incubated overnight at 37°C. The following day 2.52 µl of 10% Trifluoroacetic acid is added and the sample is incubated at room temperature for 5 minutes. Afterwards the solution is centrifuged at 16,200 rcf for 30 minutes and the supernatant is transferred to a HPLC-vial for analysis.
Mass spectrometry analysis of tryptic peptides was conducted on a 6460 Triple Quad LC/MS system (Agilent Technologies, USA). From the samples prepared according to the protocol above 10 µl were injected on to an ACQUITY UPLC® Peptide CSH™ C18 Column (Waters Corporation, USA). The peptides were separated in a gradient of buffer A (25 mM formic acid in milliQ water) and buffer B (50 mM formic acid in acetonitrile) at a flow rate of 500 µl per minute at a column temperature of 40°C. The column is equilibrated with 98:2 ratio of Buffer A to B. After injection over 20 minutes the ratio changes to 75:25 A to B after which, within 30 seconds, the ratio goes to 20:80 A to B and is held for another 30 seconds. Finally the column is flushed for 5 minutes with 98:2 A to B ratio.
EF-Tu is a constant component of the PURE system and we used its proteolytic peptide TTLTAAITTVLAK as an internal standard for variations during sample handling. All proteomics data were analysed in Skyline-daily 18.104.22.16879 (MacCoss lab, University of Washington, USA). Retention time was predicted after standard runs with the method described above using the Pierce™ Peptide Retention Time Calibration Mixture (Catalog number 88320, Thermo Scientific, USA).
Purified proteins MinD-eGFP and MinE with stock concentrations of 64 µM and 89 µM, respectively, were used as standards for quantitative LC-MS. The two proteins were mixed and a serial dilution was prepared in a buffer containing 20 mM HEPES pH 7.6, 180 mM potassium glutamate and 14 mM magnesium glutamate. Three dilution series of 10 µM, 5 µM, 2.5 µM, 1.25 µM and 0.625 µM were prepared independently of each other and treated according to the same digestion protocol as described above for the PUREfrex2.0 samples. Samples of 10 µL from two dilution rows were injected from the lowest concentration to the highest with blank measurements between every standard row. Then, the three dilutions of two biological replicates of PUREfrex2.0 reactions were injected with the lowest concentration first. The third standard row was injected last. Mass spectrometry data were analyzed in Skyline-daily as mentioned above and integrated peak intensities were plotted in OriginPro 2015 (b9.2.257, OriginLab Corporation, USA). The plotted concentrations of each peptide were fitted with ‘Instrumental’ weighting taking the variance of each value into account.
PUREfrex2.0 (GeneFrontier Corporation, Chiba) was utilized following storage and handling instructions provided by the supplier. Linear DNA constructs were added at a concentration of 5 nM for gel analysis. In co-expression reactions, minE and minD DNA templates were included at 4 nM and 8 nM, respectively, and the solution was supplemented with 1 μL of DnaK Mix (GeneFrontier Corporation). Gene expression reactions were carried out in 20-µL volume in PCR tubes for 3 h at 37 °C.
Of these reactions 5 ul were digested using the above protocol.
Created on 10/2/19, 4:10 PM