在毕赤酵母中表达的重组蛋白酶3（韦格纳抗原）具有功能活性，并能被患者血清识别。
Recombinant proteinase 3 (Wegener's antigen) expressed in Pichia pastoris is functionally active and is recognized by patient sera.
作者信息
Harmsen M C, Heeringa P, van der Geld Y M, Huitema M G, Klimp A, Tiran A, Kallenberg C G
机构信息
Department of Clinical Immunology, University of Groningen, The Netherlands.
出版信息
Clin Exp Immunol. 1997 Nov;110(2):257-64. doi: 10.1111/j.1365-2249.1997.tb08325.x.
Abstract
The open reading frame of human proteinase 3 (PR3) without the prepro-peptide was cloned and expressed in Escherichia coli (rcPR3) and in Pichia pastoris (rpPR3). The 6-histidine tagged rpPR3 was efficiently secreted into culture supernatant from which it could be purified by immobilized metal chelate chromatography. Purified rpPR3 migrated as a single 32-kD band on SDS-PAGE and harboured protease activity that could be inhibited with inhibitors specific for serine-proteases. By indirect antigen-capture ELISA using rpPR3, 60% of sera from patients with Wegener's granulomatosis bound to the recombinant product, although it was not recognized in ELISA with directly coated rpPR3.
摘要
将人蛋白酶3（PR3）去除前原肽的开放阅读框克隆出来，并在大肠杆菌（rcPR3）和毕赤酵母（rpPR3）中表达。带有6个组氨酸标签的rpPR3被有效分泌到培养上清液中，可通过固定化金属螯合层析从中纯化出来。纯化后的rpPR3在SDS-PAGE上呈现为单一的32-kD条带，并具有蛋白酶活性，该活性可被丝氨酸蛋白酶特异性抑制剂所抑制。通过使用rpPR3的间接抗原捕获ELISA检测，60%韦格纳肉芽肿患者的血清与重组产物结合，尽管在直接包被rpPR3的ELISA中未被识别。
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