Collagen, Recombinant, Human

Recombinant Human Collagen produced in E. coli is a non-glycosylated polypeptide chain a molecular mass of 96kD containing human collagen's cDNA transcribed reversely from mRNA. Amino Acid Sequence: The sequence of the first 15 N-terminal amino acids was determined and was found to be MDPVVLQRRDWENPG. Amino Acid %: Asp 0.39; Thr 0.16; Ser 5.70; Pro 18.87; Gly 29.17; Ala 13.38; Val 0.71; Met 0.18; Ile 0.16; Leu 8.04; Tyr 0.90; Phe 0.15; Lys 0.27; His 0.15; Arg 0.03; Gln 17.40; Hydro Pro 4.34. Biological Activity: 1. Cultivation of BHK-21 and 2BS cell line BHK-21 or 2BS cells were plated onto the bottom of the 96-well cell culture plates at the density of 1x10e3cells/well, respectively, with 0.1ml RPMI1640 contained 10% fetal bovine serum in the well. BHK-21 or 2BS cells were incubated in standard conditions (at 37 degrees C with 5% CO2 in a humidified incubator) for 24 hours. 2. Biochemical activity analysis of Human-like Collagen) Complete culture media was removed after 24 hours, rHuCollagen was added at various concentrations: 0.01%, 0.03%, 0.05%, 0.07%, 0.09%, 0.10%, 0.11%, 0.13% and a complete medium was used as blank control. Cells were incubated in standard conditions (at 37 degrees C with 5% CO2 in a humidified incubator). The morphology of cell was observed by reverse microscope after 24hr, 48hr, 72hr, 96hr, and 120hr. Thereafter, the viable cell count was determined by a colorimetric method using MTT (3-4,5-dimethylthiazol-2yl-2, 5-diphenyl tetrazolium bromide) after 24hr, 48hr, 72hr, 96hr and 120hr. A490 was evaluated by ELISA reader. 3. Results The results demonstrate that rHuCollagen has no significant effect on cell morphology. MTT analysis shows that rHuCollagen has remarkable effect on cell proliferation when its concentration is higher than 0.03%. Cell proliferation was improved by 40% to 60% compared to control. Endotoxin: <0.1ng/ug (IEU/ug) of Recombinant Human Collagen. Reconstitution: Reconstitute in sterile ddH2O to a concentration of not less than 0.1mg/ml, which can then be further diluted to other aqueous solutions. Storage and Stability: Store powder dessicated at -20C. Liquid can be stored at 4C for 2-7 days, long term at -20C. For long term it is recommended to add a carrier protein (0.1% HSA or BSA). Aliquot to prevent repeated freeze-thaw cycles.