引入非天然氨基酸胶原蛋白表达及交联成键的优化

Optimization of unnatural amino acid incorporation in collagen and the cross-linking through thioether bond

摘要:

微生物重组表达胶原蛋白来源清洁，同时具有序列设计灵活和高产量高纯度等优点，作为生物材料在组织工程等领域具有广泛的应用前景。然而如何促进重组胶原分子交联，使其形成更加稳定的空间结构是设计重组胶原纳米材料需要克服的难点。文中通过双质粒系统将非天然氨基酸O-(2-溴乙基)-酪氨酸引入细菌胶原蛋白序列中，并对其发酵条件进行优化，结果表明在25 ℃下，以终浓度为0.5 mmol/L的IPTG和0.06%的阿拉伯糖诱导24 h可以获得高纯度含非天然氨基酸的胶原蛋白。将含非天然氨基酸的胶原蛋白与含半胱氨酸的胶原蛋白在pH为9.0的NH4HCO3缓冲液中进行交联，形成了最大分子粒径可达1 μm的聚集体，为重组胶原蛋白生物材料的设计提供了新思路。

Abstract:

The source of recombinant collagen is clean, and it has the advantages of flexible sequence design, high yield and high purity, so it has a wide application prospect as biomaterials in tissue engineering and other fields. However, how to promote the cross-linking of recombinant collagen molecules and make them form a more stable spatial structure is the difficulty to be overcome in the design of recombinant collagen nanomaterials. Unnatural amino acid O-(2-bromoethyl)-tyrosine was incorporated into collagen by two-plasmid expression system. The results showed that high-purity collagen incorporated with unnatural amino acid could be obtained by induction with final concentration of 0.5 mmol/L IPTG and 0.06% arabinose at 25 °C for 24 hours. The intermolecular cross-linking through thioether bond was formed between collagen molecule incorporated with unnatural amino acid and collagen molecule with cysteine mutation in pH 9.0 NH4HCO3 buffer, which formed aggregates with the largest molecular size up to 1 micrometre. The results pave the way for the design of recombinant collagen biomaterials.