The expression, characterization, and crystallization of wild-type and selenomethionyl human chorionic gonadotropin.
Although the glycoprotein hormone hCG was crystallized over 4 yr ago, it is only now that three-dimensional structural information is available. This manuscript reports the method for successful production of modified expressed hormone, the characteristics of the crystallized protein, and unexpected observations during the crystallization process. Two different routes of solution to the structure of hCG were followed. The first was based on the traditional method of heavy atom isomorphous replacement, and the second was the more novel method of expressing the protein with selenomethionine substituting for methionine and applying multiwavelength anomalous diffraction analysis. Selenomethionyl hCG was employed to successfully grow the crystals used for the solution of the structure of hCG after partial deglycosylation by hydrogen fluoride (HF) treatment. The selenomethionyl hCG proved to be more hydrophobic than the expressed form of native hCG. Furthermore, expressed forms of hCG that were deglycosylated by HF proved to be more intact and less susceptible to peptide bond cleavages during the crystallization process than the urinary form of HF-treated hCG studied previously. It was found that addition of reducing agent during the crystallization period was necessary for the growth of crystals of HF-treated selenomethionyl hCG suitable for diffraction studies. Growth of crystals of HF-treated expressed hCG were accelerated by the addition of dithiothreitol, but would successfully grow without reductant. HPLC analysis of the HF-treated hormones before and during the crystallization process was used to identify alterations in the molecules, including oxidation and aggregation, both of which may affect the growth of crystals.
Lustbader
J W
JW
Department of Medicine, Columbia University, New York, New York 10032.
Wu
H
H
Birken
S
S
Pollak
S
S
Gawinowicz Kolks
M A
MA
Pound
A M
AM
Austen
D
D
Hendrickson
W A
WA
Canfield
R E
RE
D000818:Animals; D016466:CHO Cells; D006063:Chorionic Gonadotropin / Q000096:biosynthesis* / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003460:Crystallization; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis; D012645:Selenomethionine / Q000031:analogs & derivatives* / Q000737:chemistry
D006063:Chorionic Gonadotropin; D011994:Recombinant Proteins; C068857:selenomethionyl choriogonadotropin; D012645:Selenomethionine