CHEMICAL SYNTHESIS, PURIFICATION, AND CHARACTERIZATION OF 2 INFLAMMATORY PROTEINS, NEUTROPHIL ACTIVATING PEPTIDE-1 (INTERLEUKIN-8) AND NEUTROPHIL ACTIVATING PEPTIDE-2
CHEMICAL SYNTHESIS, PURIFICATION, AND CHARACTERIZATION OF 2 INFLAMMATORY PROTEINS, NEUTROPHIL ACTIVATING PEPTIDE-1 (INTERLEUKIN-8) AND NEUTROPHIL ACTIVATING PEPTIDE-2
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DOI：
10.1021/bi00226a021
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发表时间：
1991-03-26
期刊：
影响因子：
2.9
通讯作者：
AEBERSOLD, R
中科院分区：
文献类型：
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作者：
CLARKLEWIS, I;MOSER, B;AEBERSOLD, R
两个最近确定的促炎蛋白，即中性粒细胞激活肽1（NAP-1）[也称为白细胞介素-8（IL-8）]和NAP-2，化学合成，纯化，并表征。 完全保护的NAP-1/IL-8（72个残基）和NAP-2（70个残基）的肽链组装通过自动化固相方法与平均逐步产率为99.5%和99.3%，导致在70%和62%的总链组装产率，分别。 脱保护得到粗产物，通过空气氧化使其折叠，并通过两个循环的反相高压液相色谱法纯化，得到27 mg NAP-1/IL-8和22 mg NAP-2。 通过反相高压液相色谱和等电聚焦建立纯度，并通过使用质谱和Edman测序方法验证纯化产物的一级结构。 合成和重组NAP-1/IL-8对人中性粒细胞同样有效，如通过测量细胞溶质游离钙、弹性蛋白酶释放和趋化性的诱导所确定的。 合成的NAP-2在弹性蛋白酶释放和钙动员测定中等同于纯化的天然NAP-2，但其作为趋化性刺激物的效力始终较低（3-5倍），这可能表明天然制剂中存在额外的趋化性组分。 结果表明，通过化学合成，这些细胞因子可以以适合于进一步结构分析以及体内和体外功能研究的纯度和量获得。 快速生成具有明确一级结构的类似物的能力表明，这将是深入研究该炎症细胞因子家族内结构-功能关系的首选方法。
Two recently identified pro-inflammatory proteins, namely, neutrophil activating peptide 1 (NAP-1) [also termed interleukin-8 (IL-8)] and NAP-2, were chemically synthesized, purified, and characterized. The fully protected NAP-1/IL-8 (72 residues) and NAP-2 (70 residues) peptide chains were assembled by automated solid-phase methods with average stepwise yields of 99.5 and 99.3%, resulting in overall chain assembly yields of 70 and 62%, respectively. Deprotection resulted in crude products, which were allowed to fold by air oxidation, and were purified by two cycles of reverse-phase high-pressure liquid chromatography, yielding 27 mg of NAP-1/IL-8 and 22 mg of NAP-2. Purity was established by reverse-phase high-pressure liquid chromatography and isoelectric focusing, and the primary structures of the purified products were verified by using mass spectrometry and Edman sequencing methods. Synthetic and recombinant NAP-1/IL-8 were equally active on human neutrophil granulocytes as determined by measuring the induction of cytosolic free calcium, elastase release, and chemotaxis. Synthetic NAP-2 was equivalent to purified natural NAP-2 in the elastase release and calcium mobilization assays, but it was consistently less potent (3-5-fold) as a stimulus of chemotaxis, perhaps indicative of additional chemotactic components in the natural preparation. The results indicate that by chemical synthesis these cytokines can be obtained in purity and quantities suitable for further structural analysis, as well as functional studies both in vivo and in vitro. The ability to rapidly generate analogues with unambiguous primary structure suggests that this will be the method of choice for an in-depth study of structure-function relationships within this family of inflammatory cytokines.