A novel system for continuous protein refolding and on-line capture by expanded bed adsorption.
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
Standard
A novel system for continuous protein refolding and on-line capture by expanded bed adsorption. / Ferré, Henrik; Ruffet, Emmanuel; Nielsen, Lise-Lotte B; Nissen, Mogens Holst; Hobley, Timothy J; Thomas, Owen R T; Buus, Søren.
I: Protein Science, Bind 14, Nr. 8, 2005, s. 2141-53.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
Harvard
APA
Vancouver
Author
Bibtex
}
RIS
TY - JOUR
T1 - A novel system for continuous protein refolding and on-line capture by expanded bed adsorption.
AU - Ferré, Henrik
AU - Ruffet, Emmanuel
AU - Nielsen, Lise-Lotte B
AU - Nissen, Mogens Holst
AU - Hobley, Timothy J
AU - Thomas, Owen R T
AU - Buus, Søren
N1 - Keywords: Adsorption; Chromatography; Humans; Oxidation-Reduction; Protein Denaturation; Protein Folding; Recombinant Proteins; beta 2-Microglobulin
PY - 2005
Y1 - 2005
N2 - A novel two-step protein refolding strategy has been developed, where continuous renaturation-bydilution is followed by direct capture on an expanded bed adsorption (EBA) column. The performance of the overall process was tested on a N-terminally tagged version of human beta2-microglobulin (HAT-hbeta2m) both at analytical, small, and preparative scale. In a single scalable operation, extracted and denatured inclusion body proteins from Escherichia coli were continuously diluted into refolding buffer, using a short pipe reactor, allowing for a defined retention and refolding time, and then fed directly to an EBA column, where the protein was captured, washed, and finally eluted as soluble folded protein. Not only was the eluted protein in a correctly folded state, the purity of the HAThbeta2m was increased from 34% to 94%, and the product was concentrated sevenfold. The yield of the overall process was 45%, and the product loss was primarily a consequence of the refolding reaction rather than the EBA step. Full biological activity of HAT-hbeta2m was demonstrated after removal of the HAT-tag. In contrast to batch refolding, a continuous refolding strategy allows the conditions to be controlled and maintained throughout the process, irrespective of the batch size; i.e., it is readily scalable. Furthermore, the procedure is fast and tolerant toward aggregate formation, a common complication of in vitro protein refolding. In conclusion, this system represents a novel approach to small and preparative scale protein refolding, which should be applicable to many other proteins.
AB - A novel two-step protein refolding strategy has been developed, where continuous renaturation-bydilution is followed by direct capture on an expanded bed adsorption (EBA) column. The performance of the overall process was tested on a N-terminally tagged version of human beta2-microglobulin (HAT-hbeta2m) both at analytical, small, and preparative scale. In a single scalable operation, extracted and denatured inclusion body proteins from Escherichia coli were continuously diluted into refolding buffer, using a short pipe reactor, allowing for a defined retention and refolding time, and then fed directly to an EBA column, where the protein was captured, washed, and finally eluted as soluble folded protein. Not only was the eluted protein in a correctly folded state, the purity of the HAThbeta2m was increased from 34% to 94%, and the product was concentrated sevenfold. The yield of the overall process was 45%, and the product loss was primarily a consequence of the refolding reaction rather than the EBA step. Full biological activity of HAT-hbeta2m was demonstrated after removal of the HAT-tag. In contrast to batch refolding, a continuous refolding strategy allows the conditions to be controlled and maintained throughout the process, irrespective of the batch size; i.e., it is readily scalable. Furthermore, the procedure is fast and tolerant toward aggregate formation, a common complication of in vitro protein refolding. In conclusion, this system represents a novel approach to small and preparative scale protein refolding, which should be applicable to many other proteins.
U2 - 10.1110/ps.051396105
DO - 10.1110/ps.051396105
M3 - Journal article
C2 - 16046630
VL - 14
SP - 2141
EP - 2153
JO - Protein Science
JF - Protein Science
SN - 0961-8368
IS - 8
ER -
ID: 8724885