This method can help answer key questions in the biochemistry, and structural biology field of secreted proteins, such as protein structure determination of, by crystallography and cryo-EM. The main advantage of this technique is it produces large amount of recombinant secreted proteins with relatively low cost for protein structure determination. First, synthesize a DNA fragment containing a five prime BglII cutting site, the secretion signal sequence of interest in a multi-cloning site as outlined in the text protocol.
Next, digest four micrograms of the DNA with BglII and X1, and four micrograms of an expression vector DNA with BamH1 and X1.Mix 10 units of each restriction enzyme with the DNA in an 1X concentration reaction buffer. Incubate at 37 degrees Celsius for four hours. Add 10 microliters of a ligation reaction mixture containing 1X T4 DNA Ligase reaction buffer and five units of T4 DNA Ligase to a fresh 1.5 milliliter Eppendorf tube.
Then, add 100 nanograms of the linearized vector DNA and 400 nanograms of the digested DNA fragment. Incubate at 16 degree Celsius for 16 hours. Transform five microliters of the ligation mixture into 100 microliters of DH5 alpha competent cells and select the resulting colonies on an Ampicillin LB agar plate as outlined in the text protocol.
Pick between five and 10 colonies transferring each to two milliliters of LB medium containing 100 micrograms per milliliter ampicillin. Grow these colonies in a shaking incubator at 220 rpm in 37 degree Celsius for 16 hours. After this, use a DNA Miniprep kit to extract each plasmid DNA.
Confirm the clones by DNA sequencing with a forward sequencing primer as outlined in the text protocol. Using 100 nanograms of the construct plasmid, transform 40 microliters of DH10Bac competent cells as outlined in the text protocol. Incubate the transformation plates at 37 degrees Celsius for 48 hours.
Pick up two white colonies from each transformation plate and inoculate each colony in two milliliters of LB medium containing kanamycin, gentamycin, and tetracycline. Extract and verify the bacmid DNA as outlined in the text protocol. Aliquot each DNA into two tubes with 20 microliters being transferred to each tube.
Then store the tubes at minus 20 degree Celsius until ready to proceed. All subsequent steps must be performed aseptically. In a six-well plate, grow a monolayer of Sf9 cells in the culture media with 10%fetal bovine serum, and 100 micrograms per milliliter Penicillin-Streptomycin antibiotics at 27 degrees Celsius to 80%confluence.
For each transfection, add 100 microliters of unsupplemented Sf9 cell culture medium without antibiotics to a 1.5 milliliter centrifuge tube. Dilute 20 microliters of bacmid DNA into each tube and gently flick to mix. Next, add 100 microliters of unsupplemented Sf9 cell culture medium without antibiotics to a 1.5 milliliter tube for each transfection.
Dilute eight microliters of lipid transfection reagent into each tube, and mix by thoroughly pipetting up and down six times. Combine these two solutions and mix them by gently pipetting slowly up and down three times. Incubate for 20 to 40 minutes at room temperature.
Wash each well of the culture plate twice using three milliliters of unsupplemented Sf9 culture medium without antibiotics for each wash. Next, add 0.8 milliliters of unsupplemented Sf9 cell culture medium without antibiotics to each tube containing the lipid DNA mixture. Slowly pipet up and down three times to mix.
Aspirate the wash media from the plates, and overlay the samples with the transfection mixture. Incubate the transfected plate for five hours at 27 degrees Celsius. After this, replace the transfection media with the complete Sf9 cell culture medium which contains 10%FBS, and 100 micrograms per milliliter Penicillin-Streptomycin antibiotics.
Incubate at 27 degree Celsius for four days. Then, collect the supernatant in a sterile 15 milliliter conical tube. Spin at 1, 010 times g for five minutes to remove the cell debris.
Discard the pellet, and decant the supernatant to a clean tube. Store at four degrees Celsius for up to one year or until ready to use. To amplify each recombinant virus, first grow a monolayer of Sf9 cells on a 150 millimeter plate to 80%confluence.
Then aspirate the medium, and add two milliliters of the P0 virus to each well. Incubate for one hour in a 27 degree Celsius incubator, rocking the plate every 15 minutes to mix the medium with the cells. Next, add 25 milliliters of complete Grace's Media containing 10%FBS, and 100 micrograms per milliliter Penicillin-Streptomycin antibiotics.
Incubate the plate for three days in a 27 degree Celsius incubator to obtain the P1 passage virus. After this, collect the supernatant in a sterile 50 milliliter conical tube. Spin at 1, 010 times g for five minutes to remove the cell debris.
Decant the supernatant to a clean tube, and store at four degrees Celsius for up to one year or until ready to use. To amplify the virus from P1 to P2 passage, first grow a monolayer of Sf9 cells on a 150 millimeter plate to 90%confluence. Once the desired confluence is reached, aspirate the medium.
Add two milliliters of the P1 virus and incubate for one hour in a 27 degree Celsius incubator. Repeat the amplification steps to obtain the P2 and P3 passages of the virus. In this study, two modified pFastBac1 baculovirus expression vectors are used to express the secreted proteins with either the gp67 or the hemolin signal sequence by replacing the intrinsic signal sequence of the target gene.
The PBEC gp67 is successfully utilized to express the extracellular domains of the a-thal-leano receptor TDR. While the PBEC1 hemolin is successfully utilized to express the extracellular domain PRK3. Next, the purified proteins are concentrated to five milligrams per milliliter, and subjected to crystallization screening.
The conditions which yielded preliminary crystals for each protein are optimized until protein crystals larger than 20 micrometers are observed. While attempting this procedure, it's important to remember they examine the quality of the proteins by size exclusion chromatography, multi-angle light scattering, and other assays for non-aggregation status of the recombinant protein. Don't forget, to check the purity of the protein also by silver stain.
Following this procedure, other methods like X-ray crystallography and cryo-EM can be performed in order to answer additional questions, like the three dimensional structures of the produced protein. After its development, this technique paved in the way for researchers in the field of structural biology to explore the structural, and functional, and biochemical properties of secreted proteins from plants and in mammals. Don't forget that working with ultraviolet light to visualize the DNA bands on the agarose gel can be extremely hazardous, and precautions such as wearing UV protections, face shield, eye goggles, lab coat, should always be taken while performing this procedure.
Also while making buffers, be careful while handling strong acids and bases.
