People Like The Interaction Research And Application Of Collagen

Based on unique characteristics, collagen has been widely used in the preparation of various biological materials. In order to produce Superior mechanical biological materials, variety of expression systems was involved to express recombinant collagen. Human-like collagen (HLC) is industrially produced by using recombinant Escherichia coli and the high-cell-density fermentation technique, which has a good prospect in cosmetic, pharmaceutical field, food industry, biological material engineering etc. Self-aggregation of HLC is quite different from that of natural collagen. In order to investige of HLC, the clear structural information of HLC enables investigation of specific self-assembly of HLC in vitro at molecular level. Based on analysis of the various forces that involved, more concrete fibrosis principles can be achieved. Under different environmental factors, the interaction of HLC at the molecular level can provide data support to the innovation of separation and purification technology, optimization of concervation condition, and application in biological materials. As a result, more suitable separation and purification method of HLC can be developed and technical support can be provide for HLC using as substitute for native collagen used in biological materials and tissue engineering.Effects of temperature on self-interaction of human-like collagen (HLC) were investigated by hydrophobic interaction chromatography, calorimetric measurement, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Results show3types of interaction roles may exist between HLC molecules at3℃-50℃, which were divided into3narrower temperature ranges. In temperature range from3℃-22℃, hydrogen bonding plays key role in the formation of a gelatinous aggregate. In the range of22℃-38℃, hydrophobic bonds accompanied by hydrogen bonds are involved in the formation compact aggregates. When temperature is above38℃, the hydrophobic effect formed in the HLC monomer results in the loss of its ability to self-interact.Human-like collagen (HLC) has a special structure which enables it to interact with other biological molecules, so it can be immobilized on carrier and used for separation and purification process efficiently. Aggregation behavior of HLC molecules was investigated by using different composition of solution under different environmental factors. Then epichlorohydrin was used as crosslinking agent to immobilize HLC onto Sepharose microsphere to prepare a novel separation and purification medium. It was found that concentration of HLC has little effect on immobilization process while temperature and pH has more effect on it. Glucose and CaCl2can inhibit self-assembly of HLC. The results shows that1ml Sepharose microspheres reacted in5ml solution system (150g/L Glucose,0.3M CaCl2,3mg/ml HLC) at45℃and pH9.0can immobilize HLC on Sepharose microballoon sphere effectively.Similar to collagen extracted from animal tissue, industrially produced HLC is difficult to purify because HLC has the property of self-assembly, whereby it tends to form aggregates of different sizes. Based on this property, a purification process based on it was developed. The interactions of HLC fibrils were analyzed under different conditions. HLC was immobilized on the Sepharose C1-6B (HLC-Sepharose) by covalent crosslinking. This triggered lateral molecular packing of free HLC in a solution in a specific manner. A purification process based on hydrogen bonding was carried out. Microscopic and electrophoretic analyses were performed. Finally, an electrophoretically pure product was obtained. The purification process does not involve expensive equipment and other reagents. This "self-purification" method is feasible and simple.On the basis of amino acids analysis and sequence analysis, Chemoffice and Discovery studio software was used to analyze characteristic sequence of HLC. It was found that HLC has the similar helix to the collagen extracted from animal tissue, helix per revolution takes about3.3amino acids and glycine in the helix interior. Proline and alanine is locationed outside and can full contact with the solvent.On this basis, the pKa values, solvation energy and other related parameters of HLC molecular were simulated and found the pI of GAPGPPGAPGPAGPPGSA is5.8which is similar to the experimental results of HLC. At last, Discovery studio was used for homology modeling of the HLC. Through the comparison of HLC sequences in protein databases, closest sequence,1YOF_B, was selected as a template and characteristic sequence of HLC successful was achieved simulation modeling. The interaction behavior of HLC was also simulated. Compared with native or other recombinant collagen molecules, there are more direct hydrogen bonds exsited in HLC aggregates. This may be related to the serine in HLC.the which may contain a large number of HLC related.