The overall goal of this procedure is to residue-specifically incorporate noncanonical amino acid analogs into model proteins using a cell-free expression system and to provide a downstream analysis.This method can help answer key questions in the medical pharmaceutical and structure biology fields such as those of noncanonical amino acids in protein structure.The main advantage of this technique is that in the absence of any living host, toxic amino acids can be incorporated into model proteins.To begin the experiment, full stock solutions of the 20 canonical amino acids, or CAAs, and the previously prepared noncanonical amino acid stock solution, or ncAA, at room temperature.Once the solutions have thawed, vortex them frequently to redissolve precipitated amino acids and place the amino acids on ice.Next, transfer 1.4 milliliters of sterile doubly distilled water into a 15 milliliter centrifuge tube and place it on ice.Add 175 microliters of each amino acid stock solution, one after another, and thoroughly vortex the tube after each addition and put the solution back on ice.Upon addition of all amino acids, thoroughly vortex the tube.Incubate the tube until the solution is as clear as possible.Split the amino acid master mix solution into three equal volumes of 1.35 milliliters in 1.5 ml reaction tubes and keep them on ice.Add 50 micro liters of the 168 millimolar stock solution of the CAA that is analogous to the ncAA to the first split volume, to create the amino acid solution that contains all 20 CAAs.Thoroughly vortex the solution then place the tube back on ice.Aliquot the solution into reaction tubes and label them plus CAA.Flash freeze the aliquots in liquid nitrogen and store them at minus 80 degrees celsius.Next, add 50 microliters of sterile doubly distilled water to the second split volume of amino acid master mix to prepare an amino acid mixture that contains 19 CAAs, and thoroughly vortex the solution, then place it on ice.Aliquot the solution, and label the tubes minus CAA.Finally, add 50 microliters of the 168 millimolar stock solution of the ncAA to the last 1.35 milliliters, to prepare an amino acid mixture that contains 19 CAAs and the ncAA.Thoroughly vortex the mixture, and put it back on ice.Aliquot, label, and freeze the tubes.On ice, thaw three 30 microliter crude extract aliquots, one plus CAA aliquot, one minus CAA aliquot, and one ncAA aliquot, three energy buffers aliquots, and the vector DNA solution.Put sterile doubly distilled water on ice.Next, add 15 microliters of energy buffer and pipe it up and down, and vortex the solution gently to mix.15 microliters of the plus CAA amino acid solution aliquot, 10 microliters of 90 nanomolar vector DNA solution, and 20 microliters of sterile doubly distilled water to the 30 microliters of crude extract.Set up the negative control by adding 15 microliters of energy buffer, 15 microliters of minus CAA, 10 microliters of 90 nanomolar vector DNA solution, and 20 microliters of sterile doubly distilled water to the 30 microliters of crude extract.Proceed to the ncAA reaction by combining 15 microliters of energy buffer, 15 microliters of plus ncAA, 10 microliters of 90 nanomolar vector DNA solution, and 20 microliters of sterile doubly distilled water to the 30 microliters of crude extract.Then incubate all of the tubes.10 minutes before running the SDS-PAGE, dilute one microliter of each of the viscous cell-free reactions in four microliters of sterile doubly distilled water in reaction tubes.Thoroughly vortex, and shortly spin the liquids down with a mini centrifuge.Add five microliters of 2X loading buffer to the five microliters of diluted cell-free reaction.Thoroughly vortex, and spin down the liquids with a mini centrifuge.Place the reaction tubes into a heating block and ensure the lids are properly closed.Meanwhile, transfer the running buffer into the gel electrophoresis chamber.After heating, spin the liquid down with a mini centrifuge.After the spin, shortly vortex the tubes and repeat the spinning.Transfer 15 microliters of protein standard and 10 microliters of each sample into the wells, and start the electrophoresis.Carefully extract the gel from the cassette and fix the gel for 30 minutes.Then transfer the gel into staining solution for 60 minutes.After staining, transfer the gel into de-staining solution for 60 to 120 minutes.Mix equal volumes of his-binding buffer and the cell-free reaction, and pipe up the mixture up and down.Then gently vortex the tube.Next, assemble the column system.Thoroughly vortex the stock solution of the his-affinity gel until the gel resin is completely dissolved.Using a one milliliter pipette tip, transfer 250 microliters of gel resin into the column.Centrifuge the column for five to 10 seconds at 13 to 15, 000 times G.Ensure that the gel resin is completely drained, then transfer 150 to 300 microliters of the cell-free reaction his-binding buffer mixture to the column.Centrifuge the column.Discard the flow-through, and place the column back into the collection tube.Add 250 microliters of his-wash buffer and tap and gently vortex the column to resuspend the gel resin.Centrifuge the column tube.Discard the collection tube, and place the column into a new 1.5 milliliter standard reaction tube.Add 150 microliters of elution buffer, and resuspend the gel resin with tapping and gentle vortexing.Then centrifuge the column to dilute the purified protein in the elution buffer.First remove the bottom cap from the gel filtration spin column and then take off the top cap.Place the column into a two milliliter wash tube and centrifuge the column to remove the gel storage buffer.Discard the flow-through, then add up to 400 microliters of the protein storage buffer, and centrifuge the column.Repeat this step once so that the entire protein storage buffer is loaded into the gel.Place the column into a fresh collection tube and carefully add up to 100 microliters of the solution of the purified model protein directly onto the center of the gel bed.Centrifuge the column.After the buffer exchange, place the complete 100 microliters of model protein solution into an open tube into the spinning vacuum concentrator.Close the lid of the concentrator and start the rotation at 30 degrees celsius.This SDS-PAGE displays the successful incorporation of L-canavanine, or Can, into the target protein deGFP with reference cell-free reaction has the highest deGFP expression level.The cell-free Can reaction yields a slightly lower deGFP concentration while no deGFP expression is detected in the negative control.An SDS-PAGE was performed after his-tag purification and buffer exchange of the expressed deGFP molecules.HPLC
