Rothermel E, Rolf O, Götze O, Zwirner J
Abteilung für Immunologie, Universität Gottingen, Germany.
Biochim Biophys Acta. 1997 Mar 20;1351(1-2):9-12. doi: 10.1016/s0167-4781(97)00006-7.
For bacterial expression of rat anaphylatoxin C5a, the cDNA was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using rat liver RNA and degenerate primers designed according to the published amino acid sequence [1]. Surprisingly, the amino acid sequence deduced from cDNA differed at positions 55 (N for K), 63 (K for H), 67 (E for N), 68 (S for E) and 69 (H for S) from the published sequence. The overall amino acid composition, however, was unchanged because these 5 amino acids were located at different positions compared to the published sequence. As a consequence, the proposed N-glycosylation site was absent, suggesting O-glycosylation of the mature molecule. Recombinant rat C5a with a 6 histidine tag at the N-terminus was expressed in bacteria, purified and renatured. The peptide was as potent as recombinant human C5a in eliciting lysosomal enzyme release from human granulocytes.
为了在细菌中表达大鼠过敏毒素C5a，使用大鼠肝脏RNA和根据已发表的氨基酸序列设计的简并引物，通过逆转录聚合酶链反应（RT-PCR）扩增cDNA[1]。令人惊讶的是，从cDNA推导的氨基酸序列在第55位（K被N取代）、63位（H被K取代）、67位（N被E取代）、68位（E被S取代）和69位（S被H取代）与已发表序列不同。然而，总体氨基酸组成未变，因为与已发表序列相比，这5个氨基酸位于不同位置。因此，推测的N-糖基化位点不存在，提示成熟分子进行O-糖基化。在细菌中表达、纯化并复性了在N端带有6个组氨酸标签的重组大鼠C5a。该肽在诱导人粒细胞释放溶酶体酶方面与重组人C5a一样有效。
