Proost P, Van Leuven P, Wuyts A, Ebberink R, Opdenakker G, Van Damme J
Rega Institute, University of Leuven, Belgium.
Cytokine. 1995 Feb;7(2):97-104. doi: 10.1006/cyto.1995.1013.
Monocyte chemotactic proteins 2 and 3 (MCP-2 and MCP-3) are chemokines structurally and functionally related to MCP-1. In contrast to MCP-1, they are produced in low amounts by stimulated leukocytes or tumour cells. As an alternative method to generate sufficient protein for in vitro and in vivo characterization of MCP-2 and MCP-3, we have synthesized both 76-residue chemokines using Fmoc chemistry. After automated solid phase peptide synthesis at a 0.05 to 0.25 mmol scale, and purification to homogeneity by C-8 RP-HPLC, correct disulfide bridges were formed in a mixture of oxidized and reduced glutathione. The synthesis was biochemically controlled by peptide sequencing of intermediate products and proteolytic fragments of the 76-residue chemokines and by mass analysis. Purified synthetic MCP-2 and MCP-3 coeluted and comigrated with their natural counterparts on analytical reverse phase columns and SDS-PAGE, respectively. Purified and folded MCP-2 and MCP-3 were chemotactic for monocytes at 7.5 ng/ml and 5 ng/ml, respectively. These minimal effective concentrations are comparable to those of the natural chemokines. Synthetic MCPs did not induce neutrophil chemotaxis. Automated Fmoc peptide synthesis is thus a useful method, allowing fast production of chemokines and analogues thereof.
单核细胞趋化蛋白2和3（MCP - 2和MCP - 3）是在结构和功能上与MCP - 1相关的趋化因子。与MCP - 1不同，它们由受刺激的白细胞或肿瘤细胞少量产生。作为为MCP - 2和MCP - 3的体外和体内特性鉴定生成足够蛋白质的替代方法，我们使用Fmoc化学合成了这两种76个氨基酸残基的趋化因子。在0.05至0.25 mmol规模的自动固相肽合成后，通过C - 8反相高效液相色谱法纯化至同质，在氧化型和还原型谷胱甘肽的混合物中形成了正确的二硫键。通过对76个氨基酸残基趋化因子的中间产物和蛋白水解片段进行肽测序以及质量分析，对合成过程进行生化控制。纯化后的合成MCP - 2和MCP - 3在分析型反相柱上和SDS - PAGE中分别与它们的天然对应物共洗脱和共迁移。纯化并折叠后的MCP - 2和MCP - 3分别在7.5 ng/ml和5 ng/ml时对单核细胞具有趋化作用。这些最低有效浓度与天然趋化因子的浓度相当。合成的MCP不会诱导中性粒细胞趋化。因此，自动Fmoc肽合成是一种有用的方法，能够快速生产趋化因子及其类似物。
