This detailed protocol describes a highly efficient and cost effective method of bacterial expression for recombinant MMP-3 catalytic domain in E.Coli, which can be applied to other MMP therapeutic targets. E.coli lacks a complex protein folding and post-translational processing system. This method uses the R2DP cell strain to enhance eukaryotic protein expression in E.coli.
Over-production of specific MMPs leads to several diseases like cancer, cardiovascular, and neurodegenerative diseases. Active soluble MMPs are needed for the development of MMP inhibiting therapeutics. Inoculate a single isolated colony of pET His-tag pro MMP-3 catalytic domain transformant from an LB ampicillin chloramphenicol resistant plate in five milliliters of LB ampicillin chloramphenicol resistant media at 37 degrees Celsius.
Save aliquots from each culture and prepare 40%glycerol stocks. Inoculate a one liter flask containing 500 milliliters of LB ampicillin chloramphenicol resistant medium to an optical density of 0.05 to 0.1 at 600 nanometers with the overnight culture. Measure the optical density at 600 nanometers at several time points, typically for three to four hours until it falls between 0.4 and 0.6.
Induce the cultures with one molar IPTG stock to a final concentration of one millimolar. Incubate in the 37 degree shaker for an additional three to four hours. Centrifuge the cells in 250 milliliter conical bottles at 13, 000 G and four degrees Celsius for 10 minutes, repeating until the cultures are entirely pelleted.
Add three milliliters of lysis buffer per gram of pellet and resuspend the pellet by vortexing or pipetting. Add 1.25 milliliters of 10%weight by volume sodium deoxycholate per one liter of culture. Add 10 microliters of DNase I per one liter of culture.
Shake at room temperature for 30 minutes at 150 RPM. Centrifuge for 10 minutes at 13, 000 G and four degrees Celsius. Discard the supernatant.
Resuspend the pellet in 100 milliliters per liter culture of inclusion body buffer by pipetting up and down. Keep the samples on ice during sonication to prevent overheating. Sonicate each sample for six cycles of 15 seconds, an output of five and a 50%pulse.
Centrifuge the samples for 10 minutes at 13, 000 G and four degrees Celsius. Check the pellet. If the pellet is compact, discard the supernatant.
Resuspend each pellet from a one liter culture and five milliliters of solubilization buffer. Incubate for at least 30 minutes on ice to allow the proteins to solubilize. Centrifuge the cells for 10 minutes at 13, 000 times G in four degrees Celsius.
If the pellet forms after centrifugation, pour the supernatant into a separate 50 milliliter conical tube. Resuspend the pellet in another five milliliters of solubilization buffer per one liter of culture by pipetting up and down. Centrifuge for 10 minutes at 13, 000 G and four degrees Celsius.
Next, pull the supernatants. Discard the pellet or store it at minus 80 degrees Celsius for additional sonication. Blank against HT wash buffer and record the A280 of the first wash fraction.
Repeat with other fractions until A280 approaches baseline. Immediately elute His-tagged proteins by adding five milliliters of HT elution buffer. Collect the flow-through at 0.5 to one milliliter fractions in microfuge tubes labeled HT elution.
Measure the A280 of the elution fraction. If the A280 is greater than 0.3 milligrams per milliliter, dilute the fraction with HT equilibration buffer. Submerge the eluted MMP fractions in dialysis tubing in one liter of dialysis buffer one and stir the tubing and its contents on a magnetic stir for at least eight hours at four degrees Celsius.
Transfer to one liter of dialysis buffer two and stir the tubing and its contents on a magnetic stir for at least eight hours at four degrees Celsius. Next, transfer to one liter of dialysis buffer three and stir the tubing and its contents on a magnetic stir for no less than eight hours at four degrees Celsius. Transfer the sample into an appropriate container and label them as dialized MMP.
Examine the tube for any precipitate. Per one milliliter aliquot of one milligram per milliliter MMP, add 50 microliters of 20 millimolar APMA to reach a final APMA concentration of one millimolar. If a precipitate forms, centrifuge it at maximum speed for 10 minutes at four degrees Celsius.
Store the supernatant in a 1.5 milliliter microfuge tube labeled activated MMP. Discard the precipitate into a container marked for APMA waste. SDS-PAGE was run to compare the His-tag purification elution fractions of His-tag pro MMP-3 catalytic domain from BL21 DE3 cells in R2DP cells.
The first eight elution fractions of the His-tagged MMP catalytic domain and BL21 DE3 cells showed lesser protein than those in the His-tagged MMP catalytic domain in R2DP cells. The induced, refolded, concentrated, activated, and desalted fractions of MMP3 CD and R2DP cells are shown. Upon activation, the molecular weight of the activated MMP-3 CD band is approximately 20 kilodaltons, as opposed to the His-tagged zymogen which remains at around 30 kilodaltons.
When centrifuging cell lysates, pellets may not be compact. When that occurs, sonicate more and centrifuge longer. When reconcentrating the protein, precipitation may occur.
Dissolve it in solubilization buffer and repeat the process. The procedure described here provides a detailed method for soluble expression of active human MMPs that can eventually be used to understand the molecular mechanism of MMP enzymatic activity. Alternate protein purification methods such as FPLC can be performed to purified MMP proteins.
