High level expression in Escherichia coli and purification of immunoreactive recombinant bonnet monkey zone pellucida glycoprotein-3

Citation
M. Srivastava et al., High level expression in Escherichia coli and purification of immunoreactive recombinant bonnet monkey zone pellucida glycoprotein-3, PROCESS BIO, 35(5), 2000, pp. 451-457
Citations number
23
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biotecnology & Applied Microbiology","Biochemistry & Biophysics
Journal title
PROCESS BIOCHEMISTRY
ISSN journal
1359-5113 → ACNP
Volume
35
Issue
5
Year of publication
2000
Pages
451 - 457
Database
ISI
SICI code
1359-5113(200001)35:5<451:HLEIEC>2.0.ZU;2-C
Abstract
An internal fragment (978 bp) corresponding to bonnet monkey (Macaca radiat a) zone pellucida glycoprotein-3 (r-bmZP3), excluding the signal sequence a nd the transmembrane-like domain, was cloned in frame downstream of the T5 promoter under lac operator control in a pQE-30 vector. r-bmZP3 was express ed as a polyhistidine fusion protein in Escherichia coli strain BL21(pLysS) , deficient in the ompT and ion proteases. The use of protease deficient ho st cells helped in reducing the level of premature translational terminatio n products of r-bmZP3. It was expressed intracellularly as insoluble inclus ion bodies. In shake flask culture, using Luria broth medium, the presence of 0.5% glucose enhanced the expression level of r-bmZP3. Based on informat ion derived from shake flask cultures, a high cell density fermentation pro cess was developed for large scale expression of the protein. After 10 h of fed-batch fermentation, a cell concentration of 25 g dry cell weight/l (A( 600) of 65) and around 160 mg/l of the protein was expressed as inclusion b odies. Using a metal affinity column, 50 mg of the purified r-bmZP3 was obt ained from 11 of fermentation broth as compared to 4 mg/l of shake flask cu lture. The purified protein was immunoreactive as tested by ELISA and Weste rn blot, methods. (C) 2000 Elsevier Science Ltd. All rights reserved.