Characterization of a cDNA coding for human factor X.
Characterization of a cDNA coding for human factor X.
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DOI：
10.1073/pnas.81.12.3699
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发表时间：
1984
影响因子：
11.1
通讯作者：
S. P. Leytus;D. Chung;W. Kisiel;K. Kurachi;E. Davie
中科院分区：
文献类型：
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作者：
S. P. Leytus;D. Chung;W. Kisiel;K. Kurachi;E. Davie
利用人肝脏mRNA制备的含有DNA插入物的lambda gt11 cDNA文库，用抗体筛选了人因子X（一种参与血液凝固级联中期的血浆蛋白）。从2 × 10(6)个噬菌体和菌斑中分离得到10个阳性克隆。对噬菌体中含有最大插入片段的cDNA进行了测序，结果显示该cDNA插入片段编码人因子x。该cDNA插入片段包含1137个碱基对，编码该分子的一部分轻链、连接区、重链、终止密码子、短3'非编码区和poly(a)尾部。a - t - t - a - a序列位于编码序列的3'端，比TGA的终止密码子早1个碱基对，比poly(a)尾部早14个碱基对，是一个潜在的聚腺苷化或加工的识别位点。从cDNA中推断出的氨基酸序列表明，因子X是一个单链多肽，由Arg-Lys-Arg三肽连接的轻链和重链组成。单链分子通过两个（或更多）内部肽键的裂解转化为轻链和重链。在等离子体中，这两条链通过二硫键连接在一起。编码人因子X活性位点的DNA序列与另外两种参与血液凝固的维生素k依赖性丝氨酸蛋白酶凝血酶原和因子IX高度一致。这些数据与先前发表的人因子X轻链蛋白质序列数据一起建立了血浆中成熟蛋白的完整氨基酸序列。
A lambda gt11 cDNA library containing DNA inserts prepared from human liver mRNA has been screened with an antibody to human factor X, a plasma protein participating in the middle phase of the blood coagulation cascade. Ten positive clones were isolated from 2 X 10(6) phage and plaque purified. The cDNA in the phage containing the largest insert has been sequenced and shown to code for human factor X. This cDNA insert contained 1137 base pairs coding for a portion of the light chain of the molecule, a connecting region, the heavy chain, a stop codon, a short 3' noncoding region, and a poly(A) tail. The sequence of A-T-T-A-A-A, which functions as a potential recognition site for polyadenylylation or processing, was present in the 3' end of the coding sequence and preceded the stop codon of TGA by 1 base pair and the poly(A) tail by 14 base pairs. The amino acid sequence deduced from the cDNA indicated that factor X is synthesized as a single-chain polypeptide containing the light and heavy chains connected by an Arg-Lys-Arg tripeptide. The single-chain molecule is then converted to the light and heavy chains by cleavage of two (or more) internal peptide bonds. In plasma, these two chains are linked together by a disulfide bond. The DNA sequence coding for the active site of human factor X showed a high degree of identity with prothrombin and factor IX, two other vitamin K-dependent serine proteases that participate in blood coagulation. These data along with the protein sequence data previously published for the light chain of human factor X establish the complete amino acid sequence for the mature protein present in plasma.