title: The alcohol-induced conformational changes in casein micelles: a new challenge for the purification of colostrinin.
abstract: Recent advances in protein separation technology have allowed for the isolation of whey proteins and peptides of significant biological importance. In this study, we report a novel method for isolation and purification of the neuroprotective proline-rich polypeptides, also known as Colostrinin (CLN). Although CLN was first isolated from ovine colostrum and characterized as a complex of small molecular peptides, its constituents are present also in other mammal colostrums. The previous purification protocols are very tedious, time consuming, and, due to the diverse characteristics of colostrum, also very difficult to validate. Thus, the aim of this study was to develop a simple protocol with a maximum recovery rate for CLN peptides. Here we demonstrate the two-step extraction/purification method that consists of methanol extraction and ammonium sulfate precipitation as the general principles. When compared with the original material, CLN obtained by this method shows (1) similar pattern of peptides in SDS PAGE, (2) identical amino acid analysis, characterized by high content of proline (22%), a high proportion of nonpolar amino acids, a low percentage of glycine, alanine, arginine, histidine, and no tryptophan, methionine, and cysteine residues, (3) similar pattern of HPLC profiles, and (4) its ability to induce IFN gamma and TNF alpha. More importantly, the protocol for the production of high-quality CLN can be accomplished in less than a 48 h timeframe. In addition, avoidance of excessively harsh conditions preserves the structure and biological activity of the peptides.
authors.affiliation: ReGen Therapeutics Plc., London, United Kingdom. marian.l.kruzel@uth.tmc.edu
authors.forename: Marian L
authors.initials: ML
authors.lastname: Kruzel
authors.forename: Antoni
authors.initials: A
authors.lastname: Polanowski
authors.forename: Tadeusz
authors.initials: T
authors.lastname: Wilusz
authors.forename: Agata
authors.initials: A
authors.lastname: Sokołowska
authors.forename: Magdalena
authors.initials: M
authors.lastname: Pacewicz
authors.forename: Renata
authors.initials: R
authors.lastname: Bednarz
authors.forename: Jerzy A
authors.initials: JA
authors.lastname: Georgiades
mesh_terms: D000438:Alcohols / Q000494:pharmacology*; D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D002364:Caseins / Q000187:drug effects*; D002851:Chromatography, High Pressure Liquid; D003126:Colostrum; D004591:Electrophoresis, Polyacrylamide Gel; D036341:Intercellular Signaling Peptides and Proteins; D007371:Interferon-gamma / Q000378:metabolism; D000432:Methanol / Q000737:chemistry; D008823:Micelles; D008892:Milk / Q000737:chemistry; D010455:Peptides / Q000302:isolation & purification* / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D012756:Sheep; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D000438:Alcohols; D002364:Caseins; D036341:Intercellular Signaling Peptides and Proteins; D008823:Micelles; D010455:Peptides; D014409:Tumor Necrosis Factor-alpha; C105874:colostrinine; D007371:Interferon-gamma; D000645:Ammonium Sulfate; D000432:Methanol