当前位置： SCI文献检索 > PROTEIN EXPRESSION AND PURIFICATION期刊下所有文献 > Bacterial fermentation of recombinant major wasp allergen Antigen 5 using oxygen limiting growth conditions improves yield and quality of inclusion bodies.

Bacterial fermentation of recombinant major wasp allergen Antigen 5 using oxygen limiting growth conditions improves yield and quality of inclusion bodies.

Abstract：

:A process for bacterial expression and purification of the recombinant major wasp allergen Antigen 5 (Ves v 5) was developed to produce protein for diagnostic and therapeutic applications for type 1 allergic diseases. Special attention was focused on medium selection, fermentation conditions, and efficient refolding procedures. A soy based medium was used for fermentation to avoid peptone from animal origin. Animal-derived peptone required the use of isopropyl-beta-D-thiogalactopyranoside (IPTG) for the induction of expression. In the case of soy peptone, a constitutive expression was observed, suggesting the presence of a component that mimics IPTG. Batch cultivation at reduced stirrer speed caused a reduced biomass due to oxygen limitation. However, subsequent purification and processing of inclusion bodies yielded significantly higher amount of product. Furthermore, the protein composition of the inclusion bodies differed. Inclusion bodies were denatured and subjected to diafiltration. Detailed monitoring of diafiltration enabled the determination of the transition point. Final purification was conducted using cation-exchange and size-exclusion chromatography. Purified recombinant Ves v 5 was analyzed by RP-HPLC, CD-spectroscopy, SDS-PAGE, and quantification ELISA. Up to 15 mg highly purified Ves v 5 per litre bioreactor volume were obtained, with endotoxin concentrations less than 20 EU mg(-1) protein and high comparability to the natural counterpart. Analytical results confirm the suitability of the recombinant protein for diagnostic and clinical applications. The results clearly demonstrate that not only biomass, but especially growth conditions play a key role in the production of recombinant Ves v 5. This has an influence on inclusion body formation, which in turn influences the renaturation rate and absolute product yield. This might also be true for other recombinant proteins that accumulate as inclusion bodies in Escherichia coli.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Kischnick S,Weber B,Verdino P,Keller W,Sanders EA,Anspach FB,Fiebig H,Cromwell O,Suck R

doi

10.1016/j.pep.2006.01.009

keywords:

subject

Has Abstract

pub_date

2006-06-01 00:00:00

pages

621-8

issue

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(06)00023-4

journal_volume

pub_type

杂志文章

在线工具

Bacterial fermentation of recombinant major wasp allergen Antigen 5 using oxygen limiting growth conditions improves yield and quality of inclusion bodies.