The overall goal of this study, is the robust and reproducible production of an osteogenic scaffold by site-directed and covalent coupling of a newly designed BMP2 variant by click chemistry. This method can help to overcome problems connected with commonly use techniques for the treatment of non-healing of long bone defects. Absorption or encapsulation methods require super physiological amounts of BMP2 that provoke several severe side effects.
The main advantage of this technique is to use lower amounts of the new designed BMP2 variant being covalently linked to scaffolds in a side-directed manner. Propagate a single colony overnight in 50 milliliters of LB medium with 50 micrograms per milliliter of kanamycin and 100 micrograms per milliliter of ampicillin at 37 degrees Celsius. Dilute the overnight cultures one to 20 into 800 milliliters of terrific broth medium supplemented with 50 micrograms per milliliter of kanamycin and 100 micrograms per milliliter of ampicillin.
Grow the cultures at 37 degrees Celsius until an optical density at 600 nanometers of 0.7 is reached. Then add propofol L-Lycine to a final concentration of 10 millimolar. Induce gene expression by the addition of IPTG for a final concentration of one millimolar.
Grow the culture at 37 degrees Celsius for 16 hours in an orbital shaker at 180 RPM. Following incubation, centrifuge the whole culture at 9, 000 times g for 30 minutes. Discard the supernatant and re-suspend the bacterial pellet in 30 milliliters of TBSE buffer.
Weigh an empty centrifuge beaker and transfer the re-suspended pellet into the empty beaker. Then centrifuge the re-suspended pellet at 6, 360 times g for 20 minutes. Discard the supernatant and weigh the beaker with the pellet.
Subtract the weight of the empty beaker to calculate the weight of the pellet. Then re-suspend the pellet in STE buffer. Use 200 milliliters of STE buffer for every 10 grams of pellet.
Sonicate the suspension on ice before centrifuging at 6, 360 times g for 20 minutes. Discard the supernatant and weigh the pellet. After repeating the sonication and centrifugation steps four times, re-suspend the pellet in 100 milliliters of TBS buffer.
Centrifuge at 6, 360 times g for 20 minutes. Then discard the supernatant and weigh the pellet. Re-suspend the pellet in nuclease buffer with freshly added nuclease using 10 milliliters per gram of pellet.
Incubate the suspension overnight at room temperature on a stirring plate at 30 RPM. The next day add Triton Buffer to the suspension. The volume of Triton Buffer to add corresponds to a 0.5 volume part of the suspension.
After incubating for 10 minutes at room temperature, centrifuge the suspension at 6, 360 times g for 20 minutes. Discard the supernatant and weigh the pellet. Next re-suspend the pellet in TE Buffer using eight milliliters per gram of pellet.
After centrifuging as before, discard the supernatant and weigh the pellet. Now re-suspend the pellet by adding four milliliters per gram of 25 millimolar sodium acitate, PH5, and five milliliters per gram of six molar guanidinium chloride with one millimolar DTT. Incubate the suspension overnight at four degrees Celsius on a stirring plate.
Following centrifugation at 75, 500 times g for 20 minutes, the supernatant contains the unfolded, monomeric BMP2 protein. Collect the supernatant and concentrate this extract to an optical density per milliliter of 20 using a three kilodalton molecular weight cut-off membrane in a concentrating cell. Add the concentrated monomers in single drops to the Renaturation Buffer while stirring.
Then incubate the solution at room temperature for 120 hours in the dark. Adjust the pH of the solution to 3.0 using concentrated hydrochloric acid before dialyzing the solution against one millimolar hydrochloric acid. Concentrate the dialyzed solution using a 10 kilodalton molecular weight cut-off membrane in a concentrating cell.
Next load the protein solution onto the equilibrated column, dilute fractions using a linear gradient of Buffer B, collecting two milliliter fractions. Analyze 20 microliters of each fraction by SDS Polyacrylamide gel electrophoresis followed by Coomassie Brilliant Blue staining to determine the fractions containing monomers and the fractions containing dimers. Next pull dimer containing fractions.
Dialyze the pool of dimer containing fractions overnight at four degrees Celsius against five liters of one millimolar hydrochloric acid. Concentrate the product by using a 10 kilodalton concentrating centrifugal filter unit before analyzing the final product by SDS-PAGE and Coomassie Brilliant Blue staining. Combine 20 micromolar of BMP2-K3Plk or BMP2-while type which is used as a negative control with 20 microliters of Azide activated agarose beads in Reaction Buffer at a total volume of 500 microliters.
Incubate for two hours at room temperature on a rotating mixture at 20 RPM. Stop the reaction by adding a final concentration of five millimolar EDTA. Incubate the solution at room temperature for 15 minutes on a rotating mixer at 20 RPM.
Then centrifuge the samples at 20, 000 times g for one minute at room temperature and collect the supernatants. Next wash the pellet containing the beads with 1, 000 microliters of HBS 500 Buffer. Centrifuge the sample at 20, 000 times g for one minute at room temperature and collect the supernatant.
Repeat this washing procedure twice more with HBS 500 Buffer, three times with four molar magnesium chloride, and two times with PBS, collecting the supernatant each time. Store the coupled beads in 1, 000 microliters of PBS at four degrees Celsius. Perform the ALP assay as described in the text protocol.
To perform ALP staining, culture promyoblastic C2C12 cells in DMEM with 10%fetal calf serum as well as 100 units per milliliter of Penicillin G and Streptomycin at 37 degrees Celsius in a humidified atmosphere at 5%carbon dioxide. Feed C2C12 cells at a density of 30, 000 cells per well in a 96 well microplate. After letting the cells attach overnight, remove the medium and add 20 microliters per well of PMB2-K3PLK coupled beads.
Then add 20 microliters of 0.4%low melting-point agarose in each well. Centrifuge the 96 well micro-plate at 2, 000 times g for five minutes at 20 degrees Celsius. Then add 80 microliters of the same medium.
Incubate at 37 degrees Celsius in a humidified atmosphere at 5%carbon dioxide for 72 hours. Following incubation, remove the medium being careful not to detach the solidified 0.4%agarose. Add 100 microliters per well of one step MBT-BCIP sub-straight solution.
Analyze alkaline phosphatidate staining using light microscopy immediately after the purple staining becomes apparent. Use bright field microscopy and take pictures. The bio-activity of the newly produced BMP2-K3Plk variant is validated by induction of alkaline phosphatase gene expression in C2C12 cells.
The in vitro test shows similar alkaline phosphatase gene expression levels induced by either while type BMP2 or BMP2-K3Plk. Beads coated with BMP2-K3Plk via cover catalyzed azide alkyne cyclo addition yielded fluorescents when incubated with the dilabeled BMP receptor 1A eco-domain. The binding of BMP receptor 1A to the BMP2-K3Plk confirms retention of the bioactivity after coupling.
The bioactivity of the BMP2-K3Plk functionalized beads was proved by ALP mediated staining. Staining occurred only in those cells which were in direct contact with the BMP2-K3Plk functionalized beads, confirming that the protein is indeed covalently linked to the beads and not just absorbed. If the protein would be released in the absorbed situation, a more spread out staining at larger distances would have been observed, as seen here for alkaline phosphatase staining upon treatment with soluble BMP2-K2Plk.
After watching this video you should have a good understanding of how to express and produce biologically active proteins by genetical expansion and how to covalently couple a fictionalized protein to microspheres by click chemistry reaction. Generating protein variance by genetical expansion allows, in principle, the introduction of artificial amino acid at any position of the primary protein sequence. Introducing a mutation into the BMP2 sequence can effect the refolding efficiency and can also change the protein parameters.
The BMP2-K3Plk production required a modification of the established method for the expression of while type BMP2. The produced BMP2-K3Plk showed biological activity comparable to that of the while type protein. Our approach to covalently couple the BMP2 variant to a azire fictionalized agarose beads by click chemistry could be realized with high efficiency, resulting in fictionalized beads, figuring a genetic differentiation in vitro.
Standard protection according to good laboratory practice must be assured.
