Stebeck C E, Baron G S, Beecroft R P, Pearson T W
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
Mol Biochem Parasitol. 1996 Oct 18;81(1):81-8. doi: 10.1016/0166-6851(96)02678-3.
The kinetoplastid membrane protein-11 molecule was purified from Trypanosoma brucei rhodesiense and an internal peptide sequence was obtained. This sequence information was used with cosmid library screening and polymerase chain reaction amplifications of both genomic DNA and cDNA to obtain the entire DNA sequence of the encoding gene and the corresponding translated amino acid sequence of 92 residues. The sequence showed 18% divergence from the corresponding molecule of the related kinetoplastid Leishmania donovani, including one key amino acid at position 45 which may be of functional relevance. The protein had a calculated molecular mass of 11078 Da, a pI of 6.0 and an overall net charge of -2 at physiological pH. The secondary structure of the molecule was predicted to consist of two amphipathic helices connected by a random-coil segment, and suggests that it would interact with lipid bilayers in the trypanosome cell membrane. Northern and Southern blot analyses showed that the trypanosome kinetoplastid membrane protein-11 molecule was translated from a single transcript and was transcribed from a single gene copy, thus making this molecule an attractive target for knockout mutagenesis.
从布氏罗得西亚锥虫中纯化出动质体膜蛋白11分子，并获得了其内部肽段序列。利用该序列信息，通过黏粒文库筛选以及对基因组DNA和cDNA进行聚合酶链反应扩增，得到了编码基因的完整DNA序列以及由92个残基组成的相应翻译氨基酸序列。该序列与相关动质体杜氏利什曼原虫的对应分子有18%的差异，包括第45位的一个关键氨基酸，其可能具有功能相关性。该蛋白的计算分子量为11078 Da，pI为6.0，在生理pH值下的总净电荷为-2。预测该分子的二级结构由两个两亲性螺旋通过一个无规卷曲片段连接而成，这表明它可能与锥虫细胞膜中的脂质双层相互作用。Northern和Southern印迹分析表明，锥虫动质体膜蛋白11分子由单个转录本翻译而来，且由单个基因拷贝转录，因此该分子成为基因敲除诱变的一个有吸引力的靶点。
