AKSHAY GOEL,TUSHAR JOGLEKAR,KRISHNANAND TIWARI,YOGESH Mishra,MAHIMA DATLA,NARENDER DEV MANTENA
申请号：
IN6037/CHE/2015
公开号：
IN2015CH06037A
申请日:
2015.11.09
申请国别(地区):
IN
年份:
2017
代理人:
摘要：
An industrially scalable method for the preparation of carrier proteins selected from tetanus toxoid (TT), diphtheria toxoid (DT), Cross Reactive Material 197 (CRM197), Haemophilus influenzae protein D, Neisseria outer membrane protein, Pertussis toxin (PT), Pertactin (PRN) and filamentous hemagglutinin (FHA) and the like, which comprises the steps of: a) transformation of Escherichia coli with the desired gene coding for the carrier protein using a plasmid vector, b) culturing the transformed Escherichia coli in suitable culture medium under suitable conditions, c) isolation and purification of Inclusion bodies, d) denaturation and solubilization of inclusion bodies at high pH value ranging from 9 to 14, e) followed by pH adjustment within a range of 6 to 8.5, preferably at 8 of the solubilized protein using in-line static mixer for a period of 0.1 to 200 msec, to produce refolded protein, f) intermediate purification of the refolded protein using ion exchange chromatography to obtain >90 % pure and native carrier protein and g) the semi purified protein obtained in step (f) is further purified by one or more chromatographic separation using anion exchange chromatography, hydrophobic interaction chromatography, metal & dye affinity chromatography, affinity chromatography, multimodal chromatography, hydroxyapatite chromatography and size exclusion chromatography to obtain purified carrier protein.
