Patent ID: 7074758
Filing Date: 2006-07-11
Classification: A61K,A61P,C07K,Y10S

Abstract:
1. A method of isolating a gonadotropin releasing hormone selected from the group consisting of muGnRH I (SEQ ID NO:1) and muGnRH II (SEQ ID NO:2) said method comprising the steps of: (a) homogenizing hypothalami under acidic conditions to obtain a homogenate; (b) filtering said homogenate to remove fat and cell debris; (c) adding acetone in a stepwise manner to the filtrate to obtain three precipitates AC I, AC II, and AC III; (d) estimating gonadotropin releasing hormone (GnRH) activity in each of the precipitates obtained in step (c); (e) fractionating active precipitate AC II using Molecular Exclusion Chromatography to obtain fractions SG I and SG II; (f) estimating gonadotropin releasing hormone (GnRH) activity in the fractions obtained in step (e); (g) fractionating fraction SG II using ion exchange chromatography using an anionic column to collect fractions MQ I, MQ II, MQ III, and MQ IV; (h) estimating gonadotropin releasing hormone (GnRH) activity in the fractions MQ I, MQ II, MQ III, and MQ IV obtained in step (g); (i) fractionating the fraction MQ I obtained in step (h) using ion exchange chromatography using a cationic column to obtain fractions MS I, MS II, MS III, and MS IV; (j) estimating gonadotropin releasing hormone (GnRH) activity in the fractions MS I, MS II, MS III, and MS IV obtained in step (i); (k) fractionating the MS II fraction using reverse phase chromatography to obtain fractions SRP I, SRP II, and SRP III; (l) estimating gonadotropin releasing hormone (GnRH) activity in the fractions obtained in step (k); (m) fractionating fraction MQ II obtained in step (h) using reverse phase chromatography to obtain fractions QRP I, QRP II, and QRP III; (n) calculating gonadotropin releasing hormone (GnRH) activity in the fractions obtained in step (m); and (o) identifying the QRP II and SRP II fractions of steps (m) and (k) respectively as muGnRH I and muGnRH II respectively.