Automated purification of recombinant proteins: combining high-throughput with high yield.
Protein crystallography, mapping protein interactions, and other functional genomic approaches require purifying many different proteins, each of sufficient yield and homogeneity, for subsequent high-throughput applications. To fill this requirement efficiently, there is a need to develop robust, automated, high-throughput protein expression, and purification processes. We developed and compared two alternative workflows for automated purification of recombinant proteins based on expression of bacterial genes in Escherichia coli (E. coli). The first is a filtration separation protocol in which proteins of interest are expressed in a large volume, 800 ml of E. coli cultures, then isolated by filtration purification using Ni-NTA-Agarose (Qiagen). The second is a smaller scale magnetic separation method in which proteins of interest are expressed in a small volume, 25 ml, of E. coli cultures then isolated using a 96-well purification system with MagneHis Ni2+ Agarose (Promega). Both workflows provided comparable average yields of proteins, about 8 microg of purified protein per optical density unit of bacterial culture measured at 600 nm. We discuss advantages and limitations of these automated workflows, which can provide proteins with more than 90% purity and yields in the range of 100 microg to 45 mg per purification run, as well as strategies for optimizing these protocols.
Lin
Chiann-Tso
CT
Pacific Northwest National Laboratory, Richland, WA 99352, USA.
Moore
Priscilla A
PA
Auberry
Deanna L
DL
Landorf
Elizabeth V
EV
Peppler
Terese
T
Victry
Kristin D
KD
Collart
Frank R
FR
Kery
Vladimir
V
D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002474:Cell-Free System; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D005374:Filtration; D008280:Magnetics; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D020592:Shewanella / Q000737:chemistry / Q000235:genetics; D012995:Solubility; D013010:Sonication
D001426:Bacterial Proteins; D011994:Recombinant Proteins