The overall goal of the following experiments is to efficiently produce milligram quantities of Miran Kinlin three and insect cell culture, which cannot be produced in other expression hosts. This is achieved by COT transecting, a KINLIN three transfer vector together with an engineered bao virus back mid to create a recombinant bao virus carrying the KINLIN three gene. As a second step, the virus is amplified inside insect cells to produce a sufficient quantity of virions for future steps.
Next insect cells are infected to produce large quantities of Kinlin three protein in order to allow us purification via fast liquid chromatography. Results are obtained that show functional kinlin three of high purity can be produced in milligram quantities based on SDS page analysis and size exclusion chromatography. The main advantage of this technique over existing methods like kindling three expression in bacteria is that macular virus infected insect cell expression increases recombinant protein yield by tenfold.
The SF nine cells for this procedure are cultured and maintained in suspension using SF 902 media supplemented with 100 micrograms per milliliter penicillin and 100 micrograms per milliliter streptomycin. For bao virus generation, the cells should be cultured to a density range of five times 10 to the five to one times 10 to the six cells per milliliter. To prepare the SF nine cells for transfection see approximately one times 10 to the six cells per well of a sterile six well tissue culture plate in two milliliters of SF 902 media supplemented with penicillin and streptomycin leave the SF nine cells for about 20 minutes at room temperature in the fume hood to adhere to the base of the plastic wells and thus form a monolayer recombinant balo virus generation is achieved by cot transecting plasmid, DNA and BMid DNA onto a monolayer SF nine culture.
The expression vector papine neuron firm T three has the mouse KINLIN three gene firm T three. Under the control of the P 10 BAO viral promoter possesses flanking bao viral sequences to permit recombination with the back mid and encodes a C terminal hiss six tag for downstream purification for each transfection makes one to two micrograms of purified MFI T three with 0.5 micrograms of purified B mid and 100 microliters of SF 902 SFM without antibiotics. This is solution A in a separate tube, dilute six microliters of cell effect in two reagent with 100 microliters of SF 902 SFM without antibiotics.
For each transfection reaction, a master mix can be created here for reduced liquid handling if many transfect are required. This is solution B.Mix the two solutions A and B using approximately 200 microliters of each per transfection and incubated room temperature for 20 minutes to form lipid DNA complexes after 20 minutes, dilute the lipid DNA complexes with 800 microliters per transfection of SF 902 SFM without antibiotics. Next, carefully aspirate the SF nine cell monolayer media and carefully pipettes the AB solution and media mixture on top of the SF nine monolayer.
Incubate the transfected cells in a humidified incubator at 27 degrees Celsius overnight in order to create a humidified environment to prevent the cells from drying out. Place the six well dish into a plastic litted container alongside a damp paper towel. On the following day, add an additional one milliliter of SF 900 to SFM without antibiotics To each monolayer culture, incubate cells at 27 degrees Celsius for five more days.
After five days, harvest the recombinant bao virus directly from the culture medium and transfer it to a clean centrifuge tube. Clarify any SF nine cell debris by centrifugation at 1000 Gs for five minutes. At room temperature transfer the virus, which is in the resulting super names and denoted as P one to a clean tube P one virus generation can be assumed to have an expected viral titer of one times 10 to the seven PFU per milliliter.
Although a plaque SA can be performed if needed, store the P one viral stock at four degrees Celsius in the dark until use the SF nine cells used for the amplification of recombinant balo virus in suspension should be at a density of 1.4 times 10 to the six cells per milliliter and the culture should occupy one 20th of the total flask volume. For example, 50 milliliters in a two liter flask. Generally for optimum vao virus amplification and protein production, the SF nine cell should be uniform in size and spherical.
Infect the SF nine cell culture with the P one viral stock at a multiplicity of infection or MOI of 0.1 using the following formula. Incubate the P one infected insect culture at 27 degrees Celsius with shaking at 100 RPM for 72 hours. Three days later, harvest the virus by separating the cells from the media by centrifugation at 1000 Gs for five minutes.
At room temperature transfer, the clarified virus enriched media to a clean tube. This viral stock is denoted as P two and the anticipated viral titer is two times 10 to the eight PFU per milliliter. Store this viral stock at four degrees Celsius in the dark until use, save the resulting cell pellets at negative 20 degrees Celsius for later confirmation of recombinant virus production.
To begin this procedure, grow an adequate volume of SF nine cell cultures in suspension and SF 902 s fm. Supplemented with 100 micrograms per milliliter penicillin and 100 micrograms per milliliter streptomycin. The ratio of total culture volume to flask volume should be one to five.
Incubate the suspension cultures at 27 degrees Celsius with shaking at 100 RPM until they reach a cell density of two times 10 to the six cells per milliliter. When the SF nine cultures are at a density of two times 10 to the six cells per milliliter, they're ready for balo virus infection. Supplement the cultures with a final concentration of 1%fetal bovine serum followed by the P two viral stock.
To give an MOI of one incubate the infected cultures at 27 degrees Celsius with shaking at 100 RPM 72 hours post-infection, harvest recombinant poly histamine tagged lin three expressing SF nine cells by centrifugation at 1000 GS for 20 minutes At room temperature. Store the resulting cell pellet at negative 20 degrees Celsius until use or at negative 80 degrees Celsius for long-term storage. This procedure starts with thawing on ice.
The frozen pellets of vao virus infected SF nine expressing recombinant kinlin three and Resus suspending the pellets with lysis buffer. After incubating the resuspended cells with the lysis buffer for five to 10 minutes, vortex until a homogenous resus suspension is achieved for further cell disruption. Sonicate the sample in a ice bath.
Clarify the lysate by centrifugation at 48, 000 GS for one hour at four degrees Celsius. Load the resulting super names onto a five milliliter volume hist trap column pre equilibrated with lysis buffer at four degrees Celsius at a rate of one milliliter per minute. Subsequent steps for protein elution buffer exchange and protein concentration will not be demonstrated here but are detailed in the accompanying manuscript.
Once the buffer exchange protein solution is obtained, apply it onto a pre equilibrated five milliliter volume, high trap heparin HP column using an ACTA FPLC at a rate of 0.5 milliliters per minute. The bound KINLIN three is alluded using a linear sodium chloride gradient in the same buffer increasing at a rate of 10 millimolar per milliliter recombinants histamine tags. KINLIN three is expected to elute at around 0.6 molar sodium chloride after concentrating the protein, the final step is to buffer exchange the protein using size exclusion chromatography apply the purified protein onto a SUEx S 210 30 column, purify the proteins according to size by applying buffer onto the column at a rate of 0.5 milliliters per minute.
The protein alluding from the column is fractionated and monitored using the absorbent at 280 nanometers. The success of recombinant virus generation was verified by assessing the presence of recombinant Kinlin three and the COT TRANSFECTED SF nine cells by SDS page and Western analysis as seen in this representative Western blood of six small scale SF nine cultures using an anti hiss six antibody, A clear band corresponding to a 75 kilodalton hiss tagged protein was visible in each sample once the virus had been amplified and used to infect SF nine cells in large scale experiments. Recombinant KINLIN three was partially purified by immobilized metal affinity chromatography.
The main gel image in this figure shows SDS page of nickel affinity purified recombinant murine kinlin three expressed in BAO virus infected SF nine cells absorbed protein was alluded using an ole gradient shown above the gel image. MW indicates molecular weight marker LYS is the whole cell lysate FT is the flow through WI is wash one and WII is wash two. Western blot analysis was also performed using the elution fractions to confirm the presence of the engineered his tag on the recombinant protein.
Next, the partially purified KINLIN three was further purified to near homogeneity by ion exchange chromatography using a heparin column. This panel shows the elucian profile observed at 280 nanometers in blue, displaying a single symmetrical peak eluding under a linear sodium chloride gradient indicated in green, using a sodium chloride concentration range of 0.05 molar to 1.0 molar. The bottom panel shows the results of SDS page analysis of the fractionated elution demonstrating the presence of the 75 kilodalton protein Kinlin three.
As a final step, KINLIN three purity was polished by size exclusion chromatography to remove aggregates and achieve homogeneity. This representative gel filtration elucian profile is of purified Kinlin three using a SUEx S 200 in Tris HCL pH 7.5 150 millimolar sodium chloride and one millimolar DTT at 20 degrees Celsius. Based on the elution volume, KINLIN three migrates as expected for a 75 kilodalton protein suggesting that it is predominantly monomeric.
This next figure shows results from SDS page of highly concentrated purified recombinant kinlin three labeled K three at 14.5 milligrams per milliliter. The production of milligram quantities of high purity full length mouse kinlin three and insect cell culture will allow for extensive structural studies and biochemical analysis of the protein. A thermo floor based thermo shift assay was also performed in this study to determine which buffers were stabilizing for KINLIN three.
KINLIN three was diluted into various buffers, comprising a two dimensional screen of pH versus sodium chloride concentration. The top panel is a 3D histogram of the transition temperatures and the bottom panel shows the change in transition temperature from the calculated average of 50.4 degrees Celsius. The bars are colored according to the range of temperatures to which they correspond.
KINLIN three was observed to be stable at high sodium chloride concentrations within the PA range of 7.0 to 9.0 with a consistent transition temperature of 55 degrees Celsius. It was also observed that the transition temperature of Kinlin three was approximately 55 degrees Celsius within the PA range of 7.0 to 7.5. Irrespective of sodium chloride concentration.
After watching this video, you should have a good understanding of how to produce highly pure functional recombinant kindling. Three for in-depth biophysical characterization and further investigation. Similar approaches can be used with other hard to prepare protein samples, and we recommend its application in such cases.
