Small-scale, semi-automated purification of eukaryotic proteins for structure determination.
A simple approach that allows cost-effective automated purification of recombinant proteins in levels sufficient for functional characterization or structural studies is described. Studies with four human stem cell proteins, an engineered version of green fluorescent protein, and other proteins are included. The method combines an expression vector (pVP62K) that provides in vivo cleavage of an initial fusion protein, a factorial designed auto-induction medium that improves the performance of small-scale production, and rapid, automated metal affinity purification of His8-tagged proteins. For initial small-scale production screening, single colony transformants were grown overnight in 0.4 ml of auto-induction medium, produced proteins were purified using the Promega Maxwell 16, and purification results were analyzed by Caliper LC90 capillary electrophoresis. The yield of purified [U-15N]-His8-Tcl-1 was 7.5 microg/ml of culture medium, of purified [U-15N]-His8-GFP was 68 microg/ml, and of purified selenomethione-labeled AIA-GFP (His8 removed by treatment with TEV protease) was 172 microg/ml. The yield information obtained from a successful automated purification from 0.4 ml was used to inform the decision to scale-up for a second meso-scale (10-50 ml) cell growth and automated purification. 1H-15N NMR HSQC spectra of His8-Tcl-1 and of His8-GFP prepared from 50 ml cultures showed excellent chemical shift dispersion, consistent with well folded states in solution suitable for structure determination. Moreover, AIA-GFP obtained by proteolytic removal of the His8 tag was subjected to crystallization screening, and yielded crystals under several conditions. Single crystals were subsequently produced and optimized by the hanging drop method. The structure was solved by molecular replacement at a resolution of 1.7 A. This approach provides an efficient way to carry out several key target screening steps that are essential for successful operation of proteomics pipelines with eukaryotic proteins: examination of total expression, determination of proteolysis of fusion tags, quantification of the yield of purified protein, and suitability for structure determination.
Frederick
Ronnie O
RO
The University of Wisconsin Center for Eukaryotic Structural Genomics and Department of Biochemistry, University of Wisconsin, Room 141B, 433 Babcock Drive, Madison, WI 53706, USA.
Bergeman
Lai
L
Blommel
Paul G
PG
Bailey
Lucas J
LJ
McCoy
Jason G
JG
Song
Jikui
J
Meske
Louise
L
Bingman
Craig A
CA
Riters
Megan
M
Dillon
Nicholas A
NA
Kunert
John
J
Yoon
Jung Whan
JW
Lim
Ahyoung
A
Cassidy
Michael
M
Bunge
Jason
J
Aceti
David J
DJ
Primm
John G
JG
Markley
John L
JL
Phillips
George N
GN
Fox
Brian G
BG
D000595:Amino Acid Sequence; D000818:Animals; D001331:Automation; D001483:Base Sequence; D002846:Chromatography, Affinity; D003460:Crystallization; D004587:Electrophoresis, Agar Gel / Q000379:methods; D005057:Eukaryotic Cells / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010957:Plasmids; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000302:isolation & purification*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid; D014982:Xenopus laevis
D011506:Proteins; D011518:Proto-Oncogene Proteins; C090851:TCL1A protein, human; D049452:Green Fluorescent Proteins