TY - JOUR
T1 - Maximizing recovery of native protein from aggregates by optimizing pressure treatment
AU - Lefebvre, Brian G.
AU - Gage, Matthew J.
AU - Robinson, Anne Skaja
PY - 2004/3
Y1 - 2004/3
N2 - Recovering native protein from aggregates is a common obstacle in the production of recombinant proteins. Recent reports have shown that hydrostatic pressure is an attractive alternative to traditional denature-and-dilute techniques, both in terms of yield and process simplicity. To determine the effect of process variables, we subjected tailspike aggregates to a variety of pressure-treatment conditions. Maximum native tailspike yields were obtained with only short pressure incubations (<5 min) at 240 MPa. However, some tailspike aggregates were resistant to pressure, despite multiple cycles of pressure. Extending the postpressure incubation time to 4 days improved the yield of native protein from aggregates from 19.4 ± 0.9 to 47.4 ± 19.6 μg/mL (approximately 78% yield of native trimer from nonaggregate material). The nearly exclusive conversion of monomer to trimer over the time scale of days, when combined with previous kinetic data, allows for the identification of three postpressure kinetic phases: a rapid phase consisting of structured dimer conversion to trimer (30 min), an intermediate phase consisting of monomer conversion to aggregate (100 min), and a slow phase consisting of conversion of monomer to trimer (days). Optimizing the production of structured dimer can yield the highest level of folded protein. Typical refolding additives, such as glycerol, or low-temperature incubation did not improve yields.
AB - Recovering native protein from aggregates is a common obstacle in the production of recombinant proteins. Recent reports have shown that hydrostatic pressure is an attractive alternative to traditional denature-and-dilute techniques, both in terms of yield and process simplicity. To determine the effect of process variables, we subjected tailspike aggregates to a variety of pressure-treatment conditions. Maximum native tailspike yields were obtained with only short pressure incubations (<5 min) at 240 MPa. However, some tailspike aggregates were resistant to pressure, despite multiple cycles of pressure. Extending the postpressure incubation time to 4 days improved the yield of native protein from aggregates from 19.4 ± 0.9 to 47.4 ± 19.6 μg/mL (approximately 78% yield of native trimer from nonaggregate material). The nearly exclusive conversion of monomer to trimer over the time scale of days, when combined with previous kinetic data, allows for the identification of three postpressure kinetic phases: a rapid phase consisting of structured dimer conversion to trimer (30 min), an intermediate phase consisting of monomer conversion to aggregate (100 min), and a slow phase consisting of conversion of monomer to trimer (days). Optimizing the production of structured dimer can yield the highest level of folded protein. Typical refolding additives, such as glycerol, or low-temperature incubation did not improve yields.
UR - http://www.scopus.com/inward/record.url?scp=1842763662&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=1842763662&partnerID=8YFLogxK
U2 - 10.1021/bp034221v
DO - 10.1021/bp034221v
M3 - Article
C2 - 15059011
AN - SCOPUS:1842763662
SN - 8756-7938
VL - 20
SP - 623
EP - 629
JO - Biotechnology Progress
JF - Biotechnology Progress
IS - 2
ER -