Rapid microscale enzymic semisynthesis of Epidermal Growth Factor (EGF) analogues

	Rapid microscale enzymic semisynthesis of Epidermal Growth Factor (EGF) analogues
	Nice, EC; Domagala, T; Fabri, L; Nerrie, M; Walker, F; Jorissen, RN; Burgess, AW; Cui, DF; Zhang, YS
刊名	GROWTH FACTORS
	2002
卷号	20 期号:2 页码:71-80
关键词	EGF EGF receptor structure-function enzymic semisynthesis micropreparative HPLC
通讯作者	Nice, EC (reprint author), Melbourne Tumour Biol Branch, Royal Melbourne Hosp, Ludwig Inst Canc Res, POB 2008, Melbourne, Vic 3050, Australia.,
英文摘要	Epidermal Growth Factor (EGF) is a small growth factor containing 53 amino acid residues capable of stimulating the proliferation of both mesenchymal and epithelial cells. Comparison of the amino acid sequences of EGF from several species, and related proteins that can bind to the EGF receptor (e.g. TGFalpha, VGF, heparin-binding EGF, and betacellulin), suggests that Leu(47), which is highly conserved, is important for biological function. Additionally, we have shown previously, using a combination of trypsin and carboxypeptidase Y digestion of native murine EGF, that removal of Leu(47) results in more than 100-fold decrease in both receptor binding and mitogenic activity. We now describe a micromethod for the rapid generation of C-terminally modified EGFs to investigate further the role of C-terminal residues in determining functional activity. These analogues have been generated by digesting native murine EGF with trypsin, purifying the biologically inactive, but structurally intact, EGF(1-45) core by micropreparative RP-HPLC, and then reversing the action of trypsin to couple synthetic peptides (e.g. DL, DI, DF, EL, DLLW) onto the C-terminus of the EGF(1-45) core. This enzymic semisynthesis method allows multiple derivatives to be generated rapidly from microgram quantities of EGF(1-45) in sufficient quantities for sensitive biological and physicochemical analysis. We have validated the method by regenerating EGF(1-47) from EGF(1-45) with equivalent mitogenic and receptor binding activity to EGF(1-47) generated from wild type EGF by digestion with trypsin and carboxypeptidase Y. We have also investigated the effect of substituting alternative normal or nonphysiological amino acids (e.g. allo-Ile) for Asp(46), Leu(47) or Arg(48). Even small changes in these C-terminal residues reduce the mitogenic potency of the analogue.
学科主题	Cell Biology; Endocrinology & Metabolism
类目[WOS]	Cell Biology ; Endocrinology & Metabolism
关键词[WOS]	SITE-DIRECTED MUTAGENESIS ; FACTOR-ALPHA ; ESCHERICHIA-COLI ; ENZYMATIC-SYNTHESIS ; BIOLOGICAL-ACTIVITY ; MAGNETIC-RESONANCE ; CRYSTAL-STRUCTURE ; FACTOR GENE ; AMINO-ACID ; MURINE
收录类别	SCI
语种	英语
WOS记录号	WOS:000176995200002
内容类型	期刊论文
版本	出版稿
源URL	[http://202.127.25.143/handle/331003/2437]
专题	上海生化细胞研究所_上海生科院生化细胞研究所
推荐引用方式 GB/T 7714	Nice, EC,Domagala, T,Fabri, L,et al. Rapid microscale enzymic semisynthesis of Epidermal Growth Factor (EGF) analogues[J]. GROWTH FACTORS,2002,20(2):71-80.
APA	Nice, EC.,Domagala, T.,Fabri, L.,Nerrie, M.,Walker, F.,...&Zhang, YS.(2002).Rapid microscale enzymic semisynthesis of Epidermal Growth Factor (EGF) analogues.GROWTH FACTORS,20(2),71-80.
MLA	Nice, EC,et al."Rapid microscale enzymic semisynthesis of Epidermal Growth Factor (EGF) analogues".GROWTH FACTORS 20.2(2002):71-80.