Computational protocol: Enhanced Yield of Recombinant Proteins with Site-Specifically Incorporated Unnatural Amino Acids Using a Cell-Free Expression System

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[…] The trypsin cleavage sites in WT GFP were predicted by PeptideCutter (http://web.expasy.org/peptide_cutter/). In-gel digestion of purified GFP containing UAA, was performed using Trypsin Gold (Promega, Madison, WI) according to a modified manufacturer’s procedure. Briefly, the gel bands corresponding in size to GFP were finely sliced, washed for 5 minutes with water, 15 minutes with acetonitrile (ACN), and dried in a vacuum centrifuge. After reduction (10 mM dithiothreitol in 100 mM NH4HCO3) and alkylation (10% iodoacetamide in 100 mM NH4HCO3), dried gel slices were incubated on ice for 15–30 minutes with Trypsin gold (12.5 ng/µL) in digestion buffer (50 mM NH4HCO3, 5 mM CaCl2). Excess buffer was removed and 20–30 µL of digestion buffer without Trypsin Gold was added to the gel slices followed by incubation at 37°C overnight. Peptides were extracted from the gel slices by washing once with 25 mM NH4HCO3, ACN and 1% formic acid (FA). The samples were then dried in a vacuum centrifuge. The extracted peptides were purified and concentrated using ZipTip pipette tips (Millipore), following the manufacturer’s instruction.ESI-MS/MS analysis was performed using reverse phase nano-LC (Agilent Technology) connected directly to the LTQ XL Orbitrap EDT mass spectrometer (Thermo Electron, Wien, Austria) at the Analytical Research Services & Instrumentation Unit, BGU. The peptides were eluted with an increasing ACN gradient (Solvent A, 0.1% FA, 5% ACN; Solvent B, 0.1% FA, 80% ACN) over a period of 70 min. MS/MS spectra were acquired in a data-dependent fashion. Instrument control was performed using the Xcalibur software package (Thermo Electron).Theoretical monoisotopic masses for the peptides generated by trypsin digestion of WT GFP were predicted with PeptideMass (http://web.expasy.org/peptide_mass/), while fragmentation of the FSVSGEGEGDATYGK peptide and theoretical molecular masses of the peptide species were calculated with the MS-Product software at the ProteinProspector web service (http://prospector.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msproduct). Theoretical molecular masses for peptides containing UAA were adjusted manually. […]

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