细菌胶原蛋白制备工艺简单、成本低,在食品、医药材料等领域有着广泛的应用前景,但细菌胶原蛋白的热稳定性低,限制了其应用。该文以截短的酿脓链球菌胶原蛋白Scl2序列为研究对象,通过嵌合富脯氨酸序列,构建了表达高热稳定性重组胶原蛋白的大肠杆菌基因工程菌,并对重组菌株的发酵条件进行优化。结果表明,在诱导菌体浓度为OD600值为2.5,发酵时间为24 h、诱导剂终浓度为1.0 mmol/L、诱导温度为35 ℃时,富脯氨酸胶原蛋白产量最高。通过十二烷基硫酸钠-聚丙烯酰氨凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)和质谱鉴定,亲和纯化后获得高纯度胶原蛋白,圆二色谱实验证明所制备的胶原蛋白能正确折叠形成三股螺旋结构。在N端和C端同时嵌合富脯氨酸序列(Pro-Pro-Gly)10的胶原蛋白的热稳定性提高最大,Tm值与原始胶原蛋白Sp2B相比提高了23 ℃,为设计高热稳定性的重组胶原蛋白材料提供了基础。
Bacterial collagen has a wide application prospect in the fields of food and medical materials because of its simple preparation process and low cost, but its application is limited due to the low thermal stability. Based on the truncated Streptococcus pyogenes collagen Scl2 sequence, the recombinant Escherichia coli strains expressing designed collagen with high thermal stability were constructed by inserting proline-rich sequence, and the fermentation conditions of the recombinant strain were optimized. The results showed that the optimal fermentation conditions were as follows: the induced cell concentration was 2.5 with a fermentation time of 24 h; the concentration of IPTG was 1.0 mmol/L and the induction temperature was 35 ℃. Through SDS-PAGE and mass spectrometry identification, high purity collagen was obtained after affinity purification. Circular dichroism experiments showed that the prepared collagen could be correctly folded into triple helix structure. The thermal stability of collagen with proline-rich sequence (Pro-Pro-Gly)10 flanked at the N-and C-terminal was the highest, which is 23 ℃ higher than the original collagen protein Sp2B. This study provides a basis for the design of recombinant collagen materials with high thermal stability.
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