Chang G T, Ploos van Amstel H K, Hessing M, Reitsma P H, Bertina R M, Bouma B N
Department of Haematology, University Hospital Utrecht, The Netherlands.
Thromb Haemost. 1992 May 4;67(5):526-32.
Mouse C127 epithelioid cells were genetically engineered to produce biologically active gamma-carboxylated human protein S. A full length human protein S cDNA was cloned into a bovine papilloma virus (BPV) based shuttle vector under the transcriptional control of the Moloney murine sarcoma virus enhancer and the mouse metallothionein promoter. Stable expression was obtained in transfected C127 cells. Expression of gamma-carboxylated protein S was dependent on the presence of vitamin K in the culture medium. Protein sequence analysis showed that recombinant and plasma protein S have the same amino terminal sequence. Analysis of specific post-translationally modified amino acids shows that recombinant protein S is fully gamma-carboxylated and fully beta-hydroxylated. Immunoblotting analysis using polyclonal and monoclonal antibodies shows that recombinant protein S has a slightly higher molecular weight than plasma protein S. After N-Glycanase treatment, identical molecular weights are observed for recombinant and plasma protein S, indicating that the difference is caused by differences in the N-linked carbohydrate side chains. Recombinant protein S also demonstrates normal cofactor activity for activated protein C in a clotting assay. Binding studies with the complement component, C4b-binding protein (C4BP), shows that recombinant protein S binds to C4BP with the same apparent affinity as plasma protein S. Two variant molecules are also tested for their binding to C4BP. The first variant has a replacement of amino acid residue leu-608 by val and was designated B variant. The second variant has three alterations, at positions 609, 611 and 612 where the acidic amino acid residues asp, asp and glu were replaced by asn, asn and gln, respectively and this variant was designated C variant.(ABSTRACT TRUNCATED AT 250 WORDS)
对小鼠C127上皮样细胞进行基因工程改造，使其产生具有生物活性的γ-羧化人蛋白S。将全长人蛋白S cDNA克隆到基于牛乳头瘤病毒（BPV）的穿梭载体中，置于莫洛尼氏鼠肉瘤病毒增强子和小鼠金属硫蛋白启动子的转录控制之下。在转染的C127细胞中获得了稳定表达。γ-羧化蛋白S的表达取决于培养基中维生素K的存在。蛋白质序列分析表明，重组蛋白S和血浆蛋白S具有相同的氨基末端序列。对特定翻译后修饰氨基酸的分析表明，重组蛋白S完全γ-羧化且完全β-羟基化。使用多克隆和单克隆抗体进行的免疫印迹分析表明，重组蛋白S的分子量略高于血浆蛋白S。经N-糖苷酶处理后，重组蛋白S和血浆蛋白S的分子量相同，表明差异是由N-连接碳水化合物侧链的差异引起的。在凝血试验中，重组蛋白S对活化蛋白C也表现出正常辅因子活性。与补体成分C4b结合蛋白（C4BP）的结合研究表明，重组蛋白S与C4BP的结合亲和力与血浆蛋白S相同。还测试了两种变体分子与C4BP的结合。第一种变体是第608位氨基酸残基亮氨酸被缬氨酸取代，命名为B变体。第二种变体在第609、611和612位有三个改变，酸性氨基酸残基天冬氨酸、天冬氨酸和谷氨酸分别被天冬酰胺、天冬酰胺和谷氨酰胺取代，该变体命名为C变体。（摘要截短于250字）
