Production of biologically active complement factor H in therapeutically useful quantities.
Production of biologically active complement factor H in therapeutically useful quantities.
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产生治疗有效量的生物活性补体因子 H。
DOI：
10.1016/j.pep.2010.12.002
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发表时间：
2011-04
影响因子：
1.6
通讯作者：
Barlow, Paul N.
中科院分区：
文献类型：
--
作者：
Schmidt, Christoph Q.;Slingsby, Fern C.;Richards, Anna;Barlow, Paul N.
关键词：
人补体因子H（FH）是一种具有40个二硫键的丰富的155 kDa血浆糖蛋白，调节旁路途径补体级联反应。FH基因的突变和单核苷酸多态性易患年龄相关性黄斑变性、非典型溶血性尿毒症综合征和致密存款病。补充FH变异体可预防疾病是一个诱人的治疗前景。目前治疗性FH的来源仅限于人血浆，这突出了对重组材料的需求。以前在培养的植物、哺乳动物或昆虫细胞中的FH表达产生的蛋白量不足以进行充分表征，并且数量级低于治疗有用性。在此，FH的V62，Y 402变体已在巴斯德毕赤酵母细胞中重组产生（rFH）。密码子优化被证明是必不可少的，而酵母交配α因子肽的开发确保了分泌。因此，我们产生了数十毫克的rFH。在内切糖苷酶H消化N-连接的聚糖后，在肝素亲和树脂和阴离子交换色谱上纯化rFH（具有8个残留的N-乙酰葡糖胺部分）。全长rFH通过质谱法和Western印迹法使用针对C-末端的单克隆抗体进行验证。重组FH是一种单一的非聚集物质（通过动态光散射），在生化和生物测定中具有完全功能。产生了另一种形式的rFH，其中八个N-糖基化序列子被Asn-Gln取代消除，产生了无聚糖的产物。在这种潜在的非常高的表达系统中成功生产rFH使得生产治疗上有用的量在经济上可行。此外，在巴斯德毕赤酵母中易于遗传操作将允许产生具有增强的药代动力学和药效学特性的工程化FH版本。
Human complement factor H (FH), an abundant 155-kDa plasma glycoprotein with 40 disulphide bonds, regulates the alternative-pathway complement cascade. Mutations and single nucleotide polymorphisms in the FH gene predispose to development of age-related macular degeneration, atypical haemolytic uraemic syndrome and dense deposit disease. Supplementation with FH variants protective against disease is an enticing therapeutic prospect. Current sources of therapeutic FH are restricted to human blood plasma highlighting a need for recombinant material. Previously FH expression in cultured plant, mammalian or insect cells yielded protein amounts inadequate for full characterisation, and orders of magnitude below therapeutic usefulness. Here, the V62,Y402 variant of FH has been produced recombinantly (rFH) in Pichia pastoris cells. Codon-optimisation proved essential whilst exploitation of the yeast mating α-factor peptide ensured secretion. We thereby produced multiple 10s-of-milligram of rFH. Following endoglycosidase H digestion of N-linked glycans, rFH (with eight residual N-acetylglucosamine moieties) was purified on heparin-affinity resin and anion-exchange chromatography. Full-length rFH was verified by mass spectrometry and Western blot using monoclonal antibodies to the C-terminus. Recombinant FH is a single non-aggregated species (by dynamic light scattering) and fully functional in biochemical and biological assays. An additional version of rFH was produced in which eight N-glycosylation sequons were ablated by Asn–Gln substitutions resulting in a glycan-devoid product. Successful production of rFH in this potentially very highly expressing system makes production of therapeutically useful quantities economically viable. Furthermore, ease of genetic manipulation in P. pastoris would allow production of engineered FH versions with enhanced pharmacokinetic and pharmacodynamic properties.
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DOI：
10.4049/jimmunol.0804031
发表时间：
2009-06-01
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
--
作者：
Ferreira VP;Herbert AP;Cortés C;McKee KA;Blaum BS;Esswein ST;Uhrín D;Barlow PN;Pangburn MK;Kavanagh D
通讯作者：
Kavanagh D
DOI：
10.1073/pnas.0501536102
发表时间：
2005-05-17
影响因子：
11.1
作者：
Hageman, GS;Anderson, DH;Allikmets, R
通讯作者：
Allikmets, R
影响因子：
4.4
作者：
Ferreira, Viviana P.;Herbert, Andrew P.;Pangburn, Michael K.
通讯作者：
Pangburn, Michael K.
影响因子：
20.3
作者：
Jozsi, Mihaly;Strobel, Stefanie;Zipfel, Peter F.
通讯作者：
Zipfel, Peter F.
影响因子：
56.9
作者：
Edwards, AO;Ritter, R;Farrer, LA
通讯作者：
Farrer, LA