Automation of protein purification for structural genomics.
A critical issue in structural genomics, and in structural biology in general, is the availability of high-quality samples. The additional challenge in structural genomics is the need to produce high numbers of proteins with low sequence similarities and poorly characterized or unknown properties. 'Structural-biology-grade' proteins must be generated in a quantity and quality suitable for structure determination experiments using X-ray crystallography or nuclear magnetic resonance (NMR). The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. The purification procedure must yield a homogeneous protein and must be highly reproducible in order to supply milligram quantities of protein and/or its derivative containing marker atom(s). At the Midwest Center for Structural Genomics we have developed protocols for high-throughput protein purification. These protocols have been implemented on AKTA EXPLORER 3D and AKTA FPLC 3D workstations capable of performing multidimensional chromatography. The automated chromatography has been successfully applied to many soluble proteins of microbial origin. Various MCSG purification strategies, their implementation, and their success rates are discussed in this paper.
Kim
Youngchang
Y
Biosciences Division and Structural Biology Center, Argonne National Laboratory, 9700 S. Cass Ave., Bldg 202, Argonne, IL 60439, USA.
Dementieva
Irina
I
Zhou
Min
M
Wu
Ruiying
R
Lezondra
Lour
L
Quartey
Pearl
P
Joachimiak
Grazyna
G
Korolev
Olga
O
Li
Hui
H
Joachimiak
Andrzej
A
D001331:Automation; D002846:Chromatography, Affinity / Q000379:methods* / Q000706:statistics & numerical data; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D023281:Genomics; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012984:Software; D012995:Solubility
D011994:Recombinant Proteins