We have previously shown that human interferon α-2b (IFN) produced in Escherichia coli (E. coli) is heterogeneous at the N-terminal, with three major species (Ahsan et al., 2014). These are: (a) the direct translation product of the gene retaining the N-terminal methionine, (b) a species from which the methionyl residue has been removed by E. coli methionyl aminopeptidase to give the native interferon α-2b and (c) in which the N-terminal Cys residue of the latter contains an acetyl group. In this paper we overcome this heterogeneity, using engineered interferon derivatives with phenylalanine residue directly downstream of the N-terminal methionine (Met-Phe-IFN). This modification not only prevented the removal of the N-terminal methionine by E. coli methionyl aminopeptidase but also the subsequent N-acetylation. Critically, Met-Phe-IFN had enhanced activity in a biological assay. N-terminal stabilization was also achieved by fusing human cytochrome b₅ at the N-terminal of interferon (b₅-IFN-chimera). In this case also, the protein was more active than a reciprocal chimera with cytochrome b₅ at the C-terminal of interferon (Met-IFN-b₅-chimera). This latter protein also had a heterogeneous N-terminal but addition of phenylalanine following Met, (Met-Phe-IFN-b₅-chimera), resolved this problem and gave enhanced biological activity.

我们之前已经表明，大肠杆菌( E. coli )产生的人干扰素 α-2b (IFN)在 N 端是异质的，主要有三个种类 (Ahsan et al., 2014)。它们是：(a) 保留 N-末端甲硫氨酸的基因的直接翻译产物，(b) 甲硫氨酰残基已被大肠杆菌甲硫氨酰氨肽酶去除以产生天然干扰素 α-2b 的物种和（c )，其中后者的 N 端 Cys 残基含有乙酰基。在本文中，我们克服了这种异质性，使用在 N 末端甲硫氨酸 (Met-Phe-IFN) 下游直接带有苯丙氨酸残基的工程化干扰素衍生物。这种修饰不仅阻止了 N 末端甲硫氨酸的去除大肠杆菌的甲硫氨酰氨基肽酶也可随后进行N-乙酰化。至关重要的是，Met-Phe-IFN 在生物测定中具有增强的活性。N-末端稳定也通过在干扰素的N-末端融合人细胞色素b _{5 (b 5} -IFN-嵌合体)来实现。在这种情况下，该蛋白质也比在干扰素的 C 末端具有细胞色素 b ₅的相互嵌合体（Met-IFN-b ₅ -嵌合体）更具活性。后一种蛋白质也有异质的 N 端，但在 Met 之后添加苯丙氨酸（Met-Phe-IFN-b ₅ -嵌合体）解决了这个问题并增强了生物活性。