Yeast Flavohemoglobin from Candida norvegensis: Its Structural, Spectral, and Stability Properties

来自酵母的一种血红蛋白样蛋白，诺维假丝酵母（Candida norvegensis），由一条含有389个氨基酸残基的单一多肽链组成，但有两个不同的结构域，具有不同的功能：一个是位于n端区域的含血红素氧结合结构域，另一个是在剩余的c端区域发现的含有FAD的还原酶结构域(Iwaasa， H., Takagi， T. and Shikama， K. (1992) J. Mol. Biol. 227, 948-954；Shikama， K, Matsuoka， A.和Iwaasa， H. (1995) Int。学生物化学j。CellBiol。27日,1107 - 1115)。为了更详细地研究酵母黄血红蛋白的结构和功能，我们利用载体PMAL-c2在大肠杆菌（TB-1菌株）细胞中构建了两个表达系统。一种是针对黄假丝酵母血红蛋白的整个蛋白，另一种是针对氧结合结构域（血红素结构域）。利用Sephadex G-75凝胶过滤和deae -纤维素层析纯化重组蛋白，并根据其氧结合功能检测其光谱和稳定性。

A hemoglobin-like protein from yeast, Candida norvegensis, is composed of a single polypeptide chain with 389 amino acid residues, but of two distinct domains carrying different functions: One is a heme-containing oxygen binding domain in the N-terminal region and the other is a FAD containing reductase domain found in the remaining C-terminal region (Iwaasa, H., Takagi, T. and Shikama, K. (1992) J. Mol. Biol. 227, 948-954; Shikama, K., Matsuoka, A. and Iwaasa, H. (1995) Int. J. Biochem. CellBiol. 27, 1107-1115).To investigate the structure and function of this yeast flavohemoglobin in more detail, we have constructed two expression systems using a vector PMAL-c2 in E. coli cells (TB-1 strain). One is for the whole protein of Candida flavohemoglobin, and the other for the oxygen binding domain (heme domain) alone. The resulting recombinant proteins were then purified using gel filtration on Sephadex G-75 followed by DEAE-cellulose chromatography, and were examined for their spectral and stability properties in veiw of the oxygen-binding function.