Production of enzymatically active ketosteroid isomerase following insoluble expression in Escherichia coli.

Biotechnol Bioeng

Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, United Kingdom.

Published: November 2006

Enzymatically active Delta(5)-3-ketosteroid isomerase (KSI) protein with a C-terminus his(6)-tag was produced following insoluble expression using Escherichia coli. A simple, integrated process was used to extract and purify the target protein. Chemical extraction was shown to be as effective as homogenization at releasing the inclusion body proteins from the bacterial cells, with complete release taking less than 20 min. An expanded bed adsorption (EBA) column utilizing immobilized metal affinity chromatography (IMAC) was then used to purify the denatured KSI-(His(6)) protein directly from the chemical extract. This integrated process greatly simplifies the recovery and purification of inclusion body proteins by removing the need for mechanical cell disruption, repeated inclusion body centrifugation, and difficult clarification operations. The integrated chemical extraction and EBA process achieved a very high purity (99%) and recovery (89%) of the KSI-(His(6)), with efficient utilization of the adsorbent matrix (9.74 mg KSI-(His(6))/mL adsorbent). Following purification the protein was refolded by dilution to obtain the biologically active protein. Seventy-nine percent of the expressed KSI-(His(6)) protein was recovered as enzymatically active protein with the described extraction, purification, and refolding process. In addition to demonstrating the operation of this intensified inclusion body process, a plate-based concentration assay detecting KSI-(His(6)) is validated. The intensified process in this work requires minimal optimization for recovering novel his-tagged proteins, and further improves the economic advantage of E. coli as a host organism.

Download full-text PDF

Source
http://dx.doi.org/10.1002/bit.21043DOI Listing

Publication Analysis

Top Keywords

inclusion body
16
enzymatically active
12
insoluble expression
8
expression escherichia
8
escherichia coli
8
integrated process
8
chemical extraction
8
body proteins
8
ksi-his6 protein
8
active protein
8

Similar Publications

Background: The adoption of leadless pacemakers (LPMs) is increasing, yet the impact of body mass index (BMI) on procedural outcomes remains underexplored.

Objective: The purpose of this study was to explore the impact of BMI on in-hospital outcomes for patients receiving LPM implantation.

Methods: Data from the National Inpatient Sample from 2018-2021 were analyzed for patients older than 18 years who underwent LPM implantation, with specific inclusion and exclusion criteria applied.

View Article and Find Full Text PDF

Background: Sacrocolpopexy has become a favored treatment of pelvic organ prolapse due to its durability and efficacy. Sacrocolpopexy has not been standardized and there is no categorization scheme to facilitate communication or research efforts for the procedure. A systematic review was conducted to facilitate construction of a classification system for sacrocolpopexy based on extent of vaginal dissection described in the medical literature.

View Article and Find Full Text PDF

Medicine Meets Science: The Imperative of Scientific Research and Publishing for Physician-Scientists.

Indian J Radiol Imaging

January 2025

Department of Neurology, Division of Cerebrovascular Medicine and Neurology, National Cerebral and Cardiovascular Center (NCVC), Suita, Osaka, Japan.

Physician-scientists serve as conduits between clinical practice and scientific research, leveraging their unique expertise to improve patient care and drive medical innovation. This article highlights the indispensable role of research and publishing in promoting evidence-based practices, facilitating professional growth, and shaping public health policy. Drawing on historical and contemporary examples, I examine the challenges faced by physician-scientists, such as ethical dilemmas and declining engagement in research, particularly in resource-constrained settings.

View Article and Find Full Text PDF

Molecular profiling (MP), which involves testing tissue, blood, or other body fluids to identify biomarkers, has become increasingly important in cancer treatment. Genomic tumor sequencing, a specific type of MP, is commonly used to identify specific gene variants or proteins that can be targeted for treatment. Germline testing is also routinely recommended for certain cancers.

View Article and Find Full Text PDF

Effect of hempseed meal on health, growth performance, ruminal fermentation, and carcass traits of intact male goats.

Transl Anim Sci

December 2024

Department of Agricultural and Environmental Sciences, College of Agriculture, Environment and Nutrition Sciences, Tuskegee University, Tuskegee, AL 36088, USA.

Hempseed meal (HSM) is a potential alternative feedstuff for livestock due to its high protein content, but it has not been approved for animal feed in the United States due to safety concerns. This study was conducted to determine the effects of HSM on feed intake, growth performance, serum biochemistry, ruminal papillae morphology, ruminal fermentation profiles, and carcass characteristics of intact male goats. Thirty-six Boer × Spanish intact male goats were randomly assigned to one of four experimental diets ( = 9 goats/diet): 0%, 10%, 20%, and 30% HSM on as-fed basis.

View Article and Find Full Text PDF

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!