Modified CBD/RGD recombinant attachment factor for improving cell-attachment efficiency and the manufacturing method thereof

A method for manufacturing the modified CBD/RGD recombinant attachment factor for improving cell attachment efficiency, which includes the steps as below:

• constructing a colon bacillus E. coli. Firstly;
• enlarging the synthetic primer of:
• 2 5′ CATGCCATGG CATGCGGTCG TGGTGACAGC TGTCCAA 3′ 3′ CCACTGTCGA CAGGTTGATG AGGTTGGTAC CGTAC 5′
• with a poly chain reactor, and cutting it with a restriction enzyme of Nco I, then by mixing in the standard solution, it is grafted on the RGD sequence, which is disclosed by Z., Y. Chen, with a PT linker connecting to the C-terminal of the CBD;
• Putting the grafted primer into a model of pET32a expression plasmid, then by applying the standard CaCl2 process, transforming it into the BL21(DE3) E. coli by the connecting solution directly, and propagating, culturing it on a medium;
• Getting the plasmid DNA of a single colony, and by using the restriction enzyme coordinating with the poly chain reaction, sifting the strain having recombinant plasmid out;
• Culturing the constructed E. coli strain to be O.D. 600=0.7˜1 by the TB medium having 0.05-μg/ml Ampicillin;
• Adding 1-mM IPTG for carrying the expression out, centrifuging 7000 g of it in 4° C. for 10 minutes, and collecting the bacteria sample;
• Suspending the bacteria in a 20-mM Tris-HCl buffer solution (pH 7.4) having 0.1-mM PMSF and 1-mM EDTA;
• the additional EDTA and PMSF are capable of restraining the protease activity of the hydrolysis target protein before it is purified;
• Breaking the cell wall of the bacteria by a press, and centrifuging 10000 g of it in 4° C. for 15 minutes;
• Filtering it by a 0.45-μm membrane, then purifying it directly by passing through a Ni-chelate affinity column;
• Rinsing and balancing the column by a 20-mM Imidazole solution with a quantity of 5˜10 times of the column, so as to remove the redundant, non-bound protein;
• Rinsing the column by a 50˜100-mM Imidazole solution with a quantity of 5˜10 times of the column, so as to flush the non-specific-bound protein and the weakly-grafted protein out of the column;
• and
• Collecting the target protein out of the specific-bound affinity column by flushing it out.