PRODUCTION AND CHARACTERIZATION OF RECOMBINANT INSULIN-LIKE GROWTH FACTOR-I (IGF-I) AND POTENT ANALOGS OF IGF-I, WITH GLY OR ARG SUBSTITUTED FOR GLU3, FOLLOWING THEIR EXPRESSION IN ESCHERICHIA-COLI AS FUSION PROTEINS
PRODUCTION AND CHARACTERIZATION OF RECOMBINANT INSULIN-LIKE GROWTH FACTOR-I (IGF-I) AND POTENT ANALOGS OF IGF-I, WITH GLY OR ARG SUBSTITUTED FOR GLU3, FOLLOWING THEIR EXPRESSION IN ESCHERICHIA-COLI AS FUSION PROTEINS
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DOI：
10.1677/jme.0.0080029
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发表时间：
1992-02-01
影响因子：
3.5
通讯作者：
FRANCIS, GL
中科院分区：
文献类型：
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作者：
KING, R;WELLS, JRE;FRANCIS, GL
介绍了一种高效的胰岛素样生长因子-I(IGF-I)融合蛋白在大肠杆菌中的表达系统。融合蛋白由甲硫基猪生长激素([MET1]-，PGH)的前46个氨基酸组成的N末端延伸组成，随后是二肽Val-ASN。后两个残基在[MET1]-PGH(1-46)和IGF-I的N-末端甘氨酸之间提供了独特的羟胺敏感连接。融合蛋白的下游加工包括包涵体的分离、Asn-Gly键的裂解、还原的IGF-I肽的重折叠和纯化为均一。该表达系统还被用来产生两种IGF-I的变异体，其中Glu3被Gly或Arg取代，分别得到[Gly3]-IGF-I和[Arg3]-IGF-I。生产毫克量的IGF-I多肽是很容易实现的。用高效液相色谱法和N端序列分析鉴定了IGF-I、[Gly3]-IGF-I和[Arg3]-IGF-I的纯度。[Gly3]-IGF-I和[Arg3]-IGF-I在测定刺激大鼠L6成肌细胞蛋白质合成和DNA合成或抑制蛋白质分解方面比IGF-I更有效。这两个类似物与牛IGF结合蛋白-2的结合效果都很差，与大鼠L6成肌细胞上的IGF-I受体结合的效果略差。我们得出结论，与IGF-I相比，类似物对IGF结合蛋白的结合减少而不是受体结合的增加可能是其生物学效力更大的原因。
The development of an efficient expression system for insulin-like growth factor-I (IGF-I) in Escherichia coli as a fusion protein is described. The fusion protein consists of an N-terminal extension made up of the first 46 amino acids of methionyl porcine GH ([Met1]-, pGH) followed by the dipeptide Val-Asn. The latter two residues provide a unique hydroxylamine-sensitive link between [Met1]-PGH(1-46) and the N-terminal Gly of IGF-I. Downstream processing of the fusion proteins involved isolation of inclusion bodies, cleavage at the Asn-Gly bond, refolding of the reduced IGF-I peptide and purification to homogeneity. This expression system was also used to produce two variants of IGF-I in which Glu3 was substituted by either Gly or Arg to give [Gly3]-IGF-I and [Arg3]-IGF-I respectively. Production of milligram quantities of IGF-I peptide was readily achieved. The purity of the IGF-I, [Gly3]-IGF-I and [Arg3]-IGF-I was established by high-performance liquid chromatography and N-terminal sequence analysis. [Gly3]-IGF-I and [Arg3]-IGF-I were more potent than IGF-I in biological assays measuring stimulation of protein synthesis and DNA synthesis or inhibition of protein breakdown in rat L6 myoblasts. Both analogues bound verv poorly to bovine IGF-binding protein-2 and slightly less well than IGF-I to the type-1 receptor on rat L6 myoblasts. We conclude that reduced binding to IGF-binding proteins rather than increased receptor binding is the likely explanation for the greater biological potency of the analogues compared with IGF-I.