TY - JOUR
T1 - Ten-Minute Protein Purification and Surface Tethering for Continuous-Flow Biocatalysis
AU - Britton, Joshua
AU - Dyer, Rebekah
AU - Majumdar, Sudipta
AU - Raston, Colin
AU - Weiss, Gregory
PY - 2017/2/20
Y1 - 2017/2/20
N2 - Nature applies enzymatic assembly lines to synthesize bioactive compounds. Inspired by such capabilities, we have developed a facile method for spatially segregating attached enzymes in a continuous-flow, vortex fluidic device (VFD). Fused Hisn-tags at the protein termini allow rapid bioconjugation and consequent purification through complexation with immobilized metal affinity chromatography (IMAC) resin. Six proteins were purified from complex cell lysates to average homogeneities of 76 %. The most challenging to purify, tobacco epi-aristolochene synthase, was purified in only ten minutes from cell lysate to near homogeneity (>90 %). Furthermore, this “reaction-ready” system demonstrated excellent stability during five days of continuous-flow processing. Towards multi-step transformations in continuous flow, proteins were arrayed as ordered zones on the reactor surface allowing segregation of catalysts. Ordering enzymes into zones opens up new opportunities for continuous-flow biosynthesis.
AB - Nature applies enzymatic assembly lines to synthesize bioactive compounds. Inspired by such capabilities, we have developed a facile method for spatially segregating attached enzymes in a continuous-flow, vortex fluidic device (VFD). Fused Hisn-tags at the protein termini allow rapid bioconjugation and consequent purification through complexation with immobilized metal affinity chromatography (IMAC) resin. Six proteins were purified from complex cell lysates to average homogeneities of 76 %. The most challenging to purify, tobacco epi-aristolochene synthase, was purified in only ten minutes from cell lysate to near homogeneity (>90 %). Furthermore, this “reaction-ready” system demonstrated excellent stability during five days of continuous-flow processing. Towards multi-step transformations in continuous flow, proteins were arrayed as ordered zones on the reactor surface allowing segregation of catalysts. Ordering enzymes into zones opens up new opportunities for continuous-flow biosynthesis.
KW - biocatalysis
KW - continuous flow
KW - multi-step transformations
KW - protein purification
KW - thin films
UR - http://www.scopus.com/inward/record.url?scp=85011024033&partnerID=8YFLogxK
U2 - 10.1002/anie.201610821
DO - 10.1002/anie.201610821
M3 - Article
SN - 1433-7851
VL - 56
SP - 2296
EP - 2301
JO - Angewandte Chemie-International Edition
JF - Angewandte Chemie-International Edition
IS - 9
ER -