NDLI: Characterization of up-regulated proteases in an industrial recombinant Escherichia coli fermentation

Content Provider	Springer Nature Link
Author	Jordan, GL Harcum, SW
Copyright Year	2002
Abstract	Proteolytic degradation of recombinant proteins is an industry-wide challenge in host organisms such as Escherichia coli. These proteases have been linked to stresses, such as the stringent and heat-shock responses. This study reports the dramatic up-regulation of protease activity in an industrial recombinant E. coli fermentation upon induction. The objective of this project was to detect and characterize up-regulated proteases due to recombinant AXOKINE® overexpression upon IPTG induction. AXOKINE® is a 22-kDa protein currently in clinical trials as a therapeutic for obesity associated with diabetes. AXOKINE® was expressed in both the soluble and inclusion body fractions in E. coli. Sodium dodecyl sulfate gelatin–polyacrylamide gel electrophoresis (SDS-GPAGE) was used to analyze the up-regulated protease activity. Western blot analysis showed degraded AXOKINE® in both the soluble and insoluble fractions. Protease inhibitors were used to characterize the proteases. The proteases were ethylenediaminetetraacetic acid (EDTA) sensitive. The protease activity increased in the presence of phenyl-methyl sulfonyl-fluoride (PMSF), a serine protease inhibitor. The incubation buffer composition was varied with respect to Mg2+ and ATP, and the protease activity was ATP independent and Mg2+ dependent. A two-dimensional electrophoresis technique was used to estimate the pI of the proteases to be between 2.9 and 4.0. Journal of Industrial Microbiology & Biotechnology (2002) 28, 74–80 DOI: 10.1038/sj/jim/7000214
Starting Page	74
Ending Page	80
Page Count	7
File Format	PDF
ISSN	13675435
Journal	Journal of Industrial Microbiology and Biotechnology
Volume Number	28
Issue Number	2
e-ISSN	14765535
Language	English
Publisher	Nature Publishing Group
Publisher Date	2002-02-01
Publisher Place	London
Access Restriction	Subscribed
Content Type	Text
Resource Type	Article
Subject	Medicine Bioengineering Applied Microbiology and Biotechnology Biotechnology

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in