Production of Enzymatically active ketosteroid isomerase following insoluble expression in Escherichia coli

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 bacteria] 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. (c) 2006 Wiley Periodicals, Inc.

Career development: A project for the 21st century

This article reports of the papers present at the International Symposium 2006 'Shaping the future: connecting career development and workforce development'. The International Symposium 2006 provided an opportunity to move the project forward by considering career development in relation to the workforce development issues of human capital, labour supply, employability skills and older workers. In addition to these specific issues, it examined the broader issues of how career development services might contribute to workforce development and the career development information base needed to support public policy making. By way of background to this special issue on the International Symposium 2006, this paper briefly examines the context and the reasons behind career development's rise to a more prominent position on the public policy arena. Following this, the process of the International Symposium 2006 that resulted in the writing of the documents contained in the special issue are briefly outlined.

The handbook of physical education

What is the current condition of the field of physical education? How has it adapted to the rise of kinesiology, sport and exercise science and human movement studies over the last thirty years? This Handbook provides an authoritative critical overview of the field and identifies future challenges and directions. The Handbook is divided in to six parts: - Perspectives and Paradigms in Physical Education Pedagogy Research; - Cross-disciplinary Contributions to Research on Physical Education; - Learners and Learning in Physical Education; - Teachers, Teaching and Teacher Education in Physical Education; - Physical Education Curriculum; - Difference and Diversity in Physical Education. This benchmark work is essential reading for educators and students in the field of physical education.