Preparation of recombinant carbohydrate deficient active analogs of human chorionic gonadotropin from insect cells.
Preparation of recombinant carbohydrate deficient active analogs of human chorionic gonadotropin from insect cells.
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从昆虫细胞中制备重组碳水化合物缺乏的人绒毛膜促性腺激素活性类似物。
DOI：
10.1080/10826069608000071
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Bahl,OP
中科院分区：
文献类型：
--
作者：
Shao,K;Bahl,OP
人绒毛膜促性腺激素（hCG）有四个N-糖基链，每个亚基中有两个。通过单克隆抗体B17柱上的免疫亲和层析，从昆虫细胞中纯化了几种缺乏一个或多个特异性N-连接糖链的类似物。通过hCGβ特异性单克隆抗体B158柱上的第二次免疫亲和层析，去除存在的hCGβ突变体的痕量（如果有的话）。在N-糖基化的共有序列-Asn × Ser/Thr-中，Asn或Thr被Gln取代，从而抑制了N-糖基化。所有类似物在High-Five昆虫细胞中过表达，表达水平在1.5至15 μg/ml之间，并且通过非还原和还原条件下的SDS-PAGE发现是均一的。它们的分子大小范围在34 k到44 k之间。所有类似物的受体结合亲和力不变，如通过使用大鼠卵巢膜的放射性受体测定所确定的。这些类似物的可用性将有助于研究特定碳水化合物链对hCG构象和体内性质的影响。
Human chorionic gonadotropin (hCG) has four N-glycosyl chains, two in each subunit. Several analogs lacking one or more specific N-linked carbohydrate chains have been purified from insect cells by immunoaffinity chromatography on a monoclonal antibody, B17, column Traces of the hCGβ mutant present, if any, were removed by a second immunoaffinity chromatography on a column of hCGβ specific monoclonal antibody, B158. N-glycosylation was inhibited by the replacement of either Asn or Thr to Gln in the consensus sequence, -Asn × Ser/Thr-, for N-glycosylation. All analogs were overexpressed in High-Five insect cells with the expression levels ranging between 1.5 to 15 μg/ml and were found homogeneous by SDS-PAGE under nonreducing and reducing conditions. Their molecular sizes ranged between 34k to 44k. The receptor binding affinity of all the analogs was unaltered as determined by radio receptor assay using rat ovarian membranes. The availability of these analogs should facilitate studies on the effect of a specific carbohydrate chain on the conformation andin vivoproperties of hCG.