截短血红蛋白在大肠杆菌中的异源表达及性质分析

Heterologous expression and characterization of truncated hemoglobin in Escherichia coli

摘要:

截短血红蛋白(truncated hemoglobin)是一种存在于细菌中的血红蛋白。解淀粉芽孢杆菌(Bacillus amyloliquefaciens)来源的截短血红蛋白YjbI氨基酸序列与高等生物来源的YjbI存在差异。为了研究异源表达后YjbI的结构与性质，本研究以大肠杆菌(Escherichia coli) BL21(DE3)为宿主，将yjbI基因序列密码子优化后连接到表达载体pET-28a(+)上，成功进行表达，之后通过His亲和标签纯化得到较高纯度的YjbI。采用SDS-PAGE、圆二色光谱分析、血红素结合率测定和氧结合能力测定等方法对纯化后的截短血红蛋白进行了分析。发现在表达时间为26 h，2% 5-氨基乙酰丙酸(5-aminolevulinic acid, ALA)添加量的条件下产量最高，YjbI表达量由122.02 mg/L提升至133.19 mg/L。圆二色光谱分析、AlphaFold3结构预测分析发现，YjbI通过形成α-螺旋结构并折叠出血红素结合位点，最终构建出完整的蛋白质三维构象。经全波长扫描和比尔-朗伯定律计算得到，添加ALA前后YjbI的血红素结合率由13.18%提升到22.78%。利用氧化还原法对氧结合能力进行测定，表明YjbI具有较高的氧气亲和力。本研究成功实现了截短血红蛋白在大肠杆菌中的异源表达，系统分析了其结构与功能特性，为微生物血红蛋白的应用提供了理论和技术基础。

Abstract:

Truncated hemoglobin is a type of hemoglobin found in bacteria. The amino acid sequence of the truncated hemoglobin YjbI derived from Bacillus amyloliquefaciens differs from those of higher organisms and has garnered significant interest due to its distinctive structural configuration and functional characteristics. In this study, Escherichia coli BL21(DE3) was used as the host for heterologous expression of yjbI with optimized codon and connected to the expression vector pET-28a(+). High-purity YjbI was obtained after purification with the His affinity tag. The purified truncated hemoglobin was analyzed by SDS-PAGE and circular dichroism assay, and its heme-binding rate and oxygen-binding capacity were determined. After optimization, the highest yield was achieved at the expression time of 26 h and 2% 5-aminolevulinic acid (ALA) addition, and the expression level of YjbI increased from 122.02 mg/L to 133.19 mg/L. Circular dichroism and AlphaFold3 structure prediction results showed that YjbI formed α-helical structures and folds to generate the heme-binding site, ultimately assembling the complete three-dimensional conformation of the protein. The results from full wavelength scanning and calculation based on the Beer-Lambert law showed that the heme-binding rate of YjbI increased from 13.18% to 22.78% after the addition of ALA. The oxygen-binding capacity was determined by the redox method, which indicated that YjbI had a high oxygen affinity. This study successfully achieved heterologous expression of truncated hemoglobin in E. coli, systematically analyzed its structural and functional characteristics, and provided a theoretical and technical basis for the application of microbial hemoglobin.