Production of selenomethionine-labeled recombinant human neutrophil collagenase in Escherichia coli.
Molecular analogs of amino acids can be incorporated into proteins. The amino acid analog selenomethionine (SeMet) has been shown to be efficiently incorporated into the proteins of growing Escherichia coli. SeMet-containing proteins are known to produce sufficiently strong anomalous scatter permitting the solution of the selenomethionyl crystal structure by multiwavelength anomalous diffraction (MAD) techniques. The recombinant protein chosen for these studies is mature, truncated neutrophil collagenase (rmNC-t). The rmNC-t protein is a monomer of 163 amino acid residues featuring one active site and two Met residues. We developed a T7 polymerase expression system allowing incorporation of SeMet into rmNC-t protein produced in E. coli. Substitution of Met with SeMet was accomplished by culturing E. coli DL41(DE3), a SeMet-tolerant strain with metA lesion, in a defined medium containing SeMet as the sole source of Met. The SeMet-labeled rmNC-t was isolated from inclusion bodies by solubilizing in urea, purified by anion column chromatography, and then refolded in the presence of Ca2+ and Zn2+. Analysis of SeMet-labeled rmNC-t demonstrated that Met replacement was 100%. Enzymatic characterization revealed no obvious differences in activity or inhibitor binding between rmNC-t and the SeMet-labeled product. We have produced pure, active SeMet-labeled rmNC-t in sufficient quantities for macromolecular crystallography studies.
Qoronfleh
M W
MW
Department of Molecular and Cellular Biology, Sterling Winthrop Pharmaceuticals Research Division, Collegeville, PA 19426-0900, USA.
Ho
T F
TF
Brake
P G
PG
Banks
T M
TM
Pulvino
T A
TA
Wahl
R C
RC
Eshraghi
J
J
Chowdhury
S K
SK
Ciccarelli
R B
RB
Jones
B N
BN
D001483:Base Sequence; D017364:Collagenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D020784:Matrix Metalloproteinase 8; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012643:Selenium / Q000378:metabolism; D012645:Selenomethionine / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D012645:Selenomethionine; D017364:Collagenases; D020784:Matrix Metalloproteinase 8; D012643:Selenium