Palmisano W A, Henzl M T
Department of Chemistry, New Mexico State University, Las Cruces 88003.
Arch Biochem Biophys. 1991 Mar;285(2):211-20. doi: 10.1016/0003-9861(91)90351-i.
The complete coding sequence of the thymus-specific parvalbumin called avian thymic hormone (ATH) has been cloned into Escherichia coli. The translated amino acid sequence was found to be identical to the sequence of map turtle parvalbumin at 90 of 108 positions. Northern blot analysis of thymic RNA indicated a transcript length of approximately 1050 bp. However, the ATH cDNA probe failed to hybridize to poly(A)+ RNA from chicken leg muscle, a further indication that avian thymic hormone is distinct from the muscle-associated parvalbumin previously isolated from chicken. Southern analysis of chicken genomic DNA suggests the presence of a single copy of the ATH gene, and the absence of hybridization between an ATH cDNA fragment and genomic DNA from rat and rabbit is confirmatory evidence that ATH expression is restricted to avian species. One of the full length ATH cDNA clones harbored an insert that lacked all 5' noncoding sequences. This cDNA was inserted without further alteration into the prokaryotic expression vector, pKK223-3. The resulting construction, which contains eleven base pairs between the Shine-Dalgarno sequence and the initiation codon, affords reasonably high levels of expression in E. coli. In most respects, recombinant ATH mimics the tissue-derived protein, retaining a similarly high affinity for Ca2+ ion (KCa = 14 +/- 5 nM). However, in contrast to ATH isolated from chicken thymus tissue, the N-terminal alanine of recombinant ATH is unacetylated. As a result, the isoelectric point is shifted upward from 4.3 to approximately 4.8.
被称为禽胸腺激素（ATH）的胸腺特异性小白蛋白的完整编码序列已被克隆到大肠杆菌中。经翻译得到的氨基酸序列在108个位置中的90个位置上与地图龟小白蛋白的序列相同。对胸腺RNA进行的Northern印迹分析表明转录本长度约为1050 bp。然而，ATH cDNA探针未能与来自鸡腿肌肉的聚腺苷酸加尾RNA杂交，这进一步表明禽胸腺激素与先前从鸡中分离出的肌肉相关小白蛋白不同。对鸡基因组DNA进行的Southern分析表明存在ATH基因的单拷贝，并且ATH cDNA片段与大鼠和兔的基因组DNA之间不存在杂交，这是ATH表达仅限于鸟类物种的确证。其中一个全长ATH cDNA克隆含有一个缺失所有5'非编码序列的插入片段。该cDNA未经进一步改造就插入到原核表达载体pKK223-3中。所得构建体在Shine-Dalgarno序列和起始密码子之间包含11个碱基对，在大肠杆菌中能提供相当高水平的表达。在大多数方面，重组ATH模拟组织来源的蛋白质，对Ca2+离子保持相似的高亲和力（KCa = 14±5 nM）。然而，与从鸡胸腺组织中分离出的ATH不同，重组ATH的N端丙氨酸未被乙酰化。结果，等电点从4.3向上移至约4.8。
