Francis G L, Ross M, Ballard F J, Milner S J, Senn C, McNeil K A, Wallace J C, King R, Wells J R
Cooperative Research Centre for Tissue Growth and Repair, CSIRO Division of Human Nutrition, Adelaide, South Australia.
J Mol Endocrinol. 1992 Jun;8(3):213-23. doi: 10.1677/jme.0.0080213.
An efficient expression system in Escherichia coli for several biologically active insulin-like growth factor-I (IGF-I) fusion peptide analogues is described. These novel IGF-I fusion protein analogues have properties that make them very useful reagents in the investigation of IGF-I action. The analogues comprise an IGF-I sequence and the first 11 amino acids of methionyl porcine growth hormone (pGH) and include [Met1]-pGH(1-11)-Val-Asn-IGF-I, which contains the authentic IGF-I sequence, and two analogues, [Met1]-pGH(1-11)-Val-Asn-[Gly3]-IGF-I and [Met1]-pGH(1-11)-Val-Asn-[Arg3]-IGF-I, where Glu-3 in the human IGF-I sequence has been replaced by Gly or Arg respectively. The three peptides are referred to as Long IGF-I, Long [Gly3]-IGF-I or Long [Arg3]-IGF-I depending on the IGF-I sequence present. Production of the purified fusion peptides was aided by folding the reduced and denatured fusion peptide sequence under conditions that gave very high yields of biologically active product. Introduction of a hydrophobic N-terminal extension peptide appears to facilitate the correct folding of the IGF-I analogues compared with that obtained previously when folding normal-length IGFs. The biological activities of the IGF-I fusion peptides were compared with authentic IGF-I and the truncated analogue, des(1-3)IGF-I. In L6 rat myoblasts, all the analogues were more potent than authentic IGF-I in their abilities to stimulate protein and DNA synthesis and inhibit protein breakdown. In H35 hepatoma cells, where the IGFs act through the insulin receptor, the Long IGF-I analogues maintained a similar potency relative to IGF-I as was observed in the L6 myoblasts. The order of biological potency in cell lines secreting IGF-binding proteins (IGFBPs) into the medium was Long [Arg3]-IGF-I-des(1-3)IGF-I greater than Long [Gly3]-IGF-I greater than Long IGF-I greater than IGF-I. In chicken embryo fibroblasts, a cell line that does not secrete detectable IGFBPs into the medium, Long [Arg3]-IGF-I, was less potent than IGF-I. Investigation of receptor and IGFBP association by these analogues reinforced our previous findings that N-terminal analogues of IGF-I show increased biological potency due to changes in the degree of their IGFBP interactions.
本文描述了一种在大肠杆菌中高效表达多种具有生物活性的胰岛素样生长因子-I（IGF-I）融合肽类似物的系统。这些新型IGF-I融合蛋白类似物具有一些特性，使其成为研究IGF-I作用时非常有用的试剂。这些类似物包含一个IGF-I序列和甲硫氨酰猪生长激素（pGH）的前11个氨基酸，包括含有天然IGF-I序列的[Met1]-pGH(1-11)-Val-Asn-IGF-I，以及两个类似物，[Met1]-pGH(1-11)-Val-Asn-[Gly³]-IGF-I和[Met1]-pGH(1-11)-Val-Asn-[Arg³]-IGF-I，其中人IGF-I序列中的Glu-3分别被Gly或Arg取代。根据所存在的IGF-I序列，这三种肽分别称为长IGF-I、长[Gly³]-IGF-I或长[Arg³]-IGF-I。通过在能产生非常高产量生物活性产物的条件下折叠还原和变性的融合肽序列，有助于纯化融合肽产量的提高。与之前折叠正常长度IGF时相比，引入疏水的N端延伸肽似乎有助于IGF-I类似物的正确折叠。将IGF-I融合肽的生物活性与天然IGF-I及截短类似物des(1-3)IGF-I进行了比较。在L6大鼠成肌细胞中，所有类似物在刺激蛋白质和DNA合成以及抑制蛋白质分解的能力方面都比天然IGF-I更强。在H35肝癌细胞中，IGF通过胰岛素受体发挥作用，长IGF-I类似物相对于IGF-I的效力与在L6成肌细胞中观察到的相似。在向培养基中分泌IGF结合蛋白（IGFBP）的细胞系中，生物效力顺序为长[Arg³]-IGF-I-des(1-3)IGF-I＞长[Gly³]-IGF-I＞长IGF-I＞IGF-I。在鸡胚成纤维细胞（一种不向培养基中分泌可检测到的IGFBP的细胞系）中，长[Arg³]-IGF-I的效力低于IGF-I。通过这些类似物对受体和IGFBP结合的研究强化了我们之前的发现，即IGF-I的N端类似物由于其与IGFBP相互作用程度的变化而显示出增强的生物效力。
