By a News Reporter-Staff News Editor at Science Letter -- Investigators discuss new findings in Chromatography. According to news reporting originating from Vienna, Austria, by NewsRx correspondents, research stated, "A reversed phase high pressure liquid chromatography method was developed for determination of in vitro refolding and cleavage kinetics for the N-pro autoprotease fusion peptide EDDIE-pep6His using a TSK Super-Octyl column with a segmented acetonitrile gradient. Self-cleaving fusion proteins such as N-pro autoprotease fusion proteins consist of the single autoprotease N-pro and a target peptide or a target protein as fusion partner."
Our news editors obtained a quote from the research from the University of Natural Resources and Applied Life Science, "Hence, three protein species are present after self-cleavage: the target peptide or protein, the single N-pro autoprotease and, in case of incomplete cleavage, residual N-pro fusion protein. Thus, for an accurate analysis the method must be standardized for three components in the presence of host cell impurities. For method validation, protein standards of EDDIE-pep6His and the single N-pro autoprotease EDDIE were prepared from inclusion bodies (IBs) by ion exchange, immobilized metal ion affinity, size exclusion, and reversed phase chromatography. A linear correlation was obtained for EDDIE-pep6His and EDDIE in the range from 95 to 730 mu g/ml with a lower limit of quantification (LLOQ) and a lower limit of detection (LLOD) of 34.5 and 11.4 mu g/ml, respectively, for EDDIE-pep6His and 39.6 and 13.1 mu g/ml, respectively, for EDDIE. Finally, a fully automated batch refolding of EDDIE-pep6His from IBs was performed to demonstrate the applicability of this method."
According to the news editors, the research concluded: "It was shown that the initial EDDIE-pep6His concentration in the refolding solution decreased from 194.3 to 83.8 mu g/ml over a refolding time of 385 min resulting in a final refolding and cleavage yield of 50%."
For more information on this research see: Autoprotease N-pro: Analysis of self-cleaving fusion protein. Journal of Chromatography A, 2013;1304():92-100. Journal of Chromatography A can be contacted at: Elsevier Science Bv, PO Box 211, 1000 Ae Amsterdam, Netherlands. (Elsevier - www.elsevier.com; Journal of Chromatography A - www.elsevier.com/wps/product/cws_home/502688)
The news editors report that additional information may be obtained by contacting M. Wellhoefer, Univ Nat Resources & Life Sci Vienna, Dept. of Biotechnol, A-1190 Vienna, Austria. Additional authors for this research include W. Sprinzl, R. Hahn and A. Jungbauer (see also Chromatography).
Keywords for this news article include: Vienna, Europe, Austria, Chromatography
Our reports deliver fact-based news of research and discoveries from around the world. Copyright 2013, NewsRx LLC