fulltext.study @t Gmail

Alternative production process strategies in E. coli improving protein quality and downstream yields

Paper ID Volume ID Publish Year Pages File Format Full-Text
35343 45087 2009 7 PDF Available
Title
Alternative production process strategies in E. coli improving protein quality and downstream yields
Abstract

Process strategies for production of recombinant rhamnulose 1-phosphate aldolase (RhuA) in Escherichia coli were found to have an important impact on downstream processing when preparing the enzyme for its use as immobilized biocatalyst. First, a continuous inducer feed was implemented in substrate limited fed-batch cultures to overexpress RhuA with a hexa histidine-tag (6xHis-tag) at its N-terminus. The final specific RhuA level was 180 mg g−1 DCW, but the final specific enzyme activity (1.7 AU mg−1 RhuA) was considerably lower than expected. Only 55% of immobilization yield was achieved when immobilized metal affinity chromatography (IMAC) was used to purify and immobilize RhuA from cellular lysate in a single step. Western blot analyses showed that only 20% of overexpressed RhuA kept the whole 6xHis-tag at the end of the culture due to partial proteolysis. Two different growth strategies improved protein quality and immobilization yield:(i)Temperature reduction to 28 °C in substrate limited operation decreased proteolysis and allowed higher specific activities, 210 mg g−1 DCW. The enzyme activity increased to 4 AU mg−1 RhuA and purification-immobilization yield to 93%.(ii)A novel fed-batch operational procedure, working at high glucose concentration was implemented. High aldolase levels, 233 mg g−1 DCW, were reached at the end of the culture. The final enzyme activity was also higher than 4 AU mg−1 RhuA, and 95% of immobilization yield was achieved.For both cases, Western blot analyses showed that 80–100% of overexpressed RhuA kept the whole 6xHis-tag at the end of the culture, confirming that recombinant protein quality had been improved.

Keywords
E. coli; Protein quality; One step purification-immobilization; Substrate in excess fed-batch cultures; Continuous inducer dosage; Aldolase
First Page Preview
Alternative production process strategies in E. coli improving protein quality and downstream yields
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us
Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 44, Issue 9, September 2009, Pages 1039–1045
Authors
, , , , , , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us