Biology; Chemistry and chemical biology; Chemical and biological engineering; Biomedical engineering
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Affinity Purification of Secreted Alkaline Phosphatase Produced by the Baculovirus Expression Vector System, F. Zhang, M.W. Wolff, D. Williams, K. Busch, S.C. Lang, D.W. Murhammer, R.J. Linhardt, Applied Biochemistry and Biotechnology, 90, 125-136, 2001.
Human secreted alkaline phosphatase (SEAP) was produced in a stably-transformed Spodoptera frugiperda Sf-9 insect cell line (Sfb4GalT) following infection with a recombinant Autographa californica multiple nuclear polyhedrovirus containing the SEAP gene under control of the polyhedrin promoter. An affinity chromatographic column prepared by linking 4-amino-benzylphosphonic acid to histidyl-expoxy-Sepharose was used to isolate SEAP from the cell supernatant following removal of cells and virus and 10-fold concentration through ultrafiltration. We found that the binding of SEAP on the affinity matrix follows the Langmuir isotherm model. In addition, either recycling SEAP sample through the column for 24 h or loading high SEAP concentrations resulted in a high-purity product. Some nonspecific binding of protein on the matrix occurred when low concentrations of SEAP sample were loaded. Finally, we found that SEAP binding occurs rapidly, i.e., within 30 min of adding the SEAP sample to the affinity matrix.;
Applied Biochemistry and Biotechnology, 90, 125-136; Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.
The Linhardt Research Labs.; The Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS);
The Linhardt Research Labs Online Collection; Rensselaer Polytechnic Institute, Troy, NY; Applied Biochemistry and Biotechnology - Part A Enzyme Engineering and Biotechnology; https://harc.rpi.edu/;