Recombinant rabbit uteroglobin expressed at high levels in E. coli forms stable dimers and binds progesterone.
In order to express uteroglobin in Escherichia coli we have constructed a DNA coding for complete mature rabbit uteroglobin by fusing genomic sequences from the second exon of the gene to an incomplete cDNA. This DNA was inserted into various positions of the polylinker cloning region of pDS expression vectors and the uteroglobin gene was expressed in E. coli by IPTG induction. Four different uteroglobin-derived proteins were produced containing 1, 3, 5 and 7 more N-terminal amino acids than the naturally occurring mature protein. The yield of soluble protein strongly increased with increasing length of the N-terminal additions. Protein and RNA analysis showed that this variation is most likely due to progressively higher translation efficiencies of the larger recombinants. UG7, the most efficiently synthesized recombinant protein, carrying seven additional N-terminal amino acids, was purified and further characterized. Like natural uteroglobin, UG7 forms a dimer and binds progesterone with an affinity indistinguishable to the natural protein. This bacterially produced protein can be used for detailed structure-function investigations of uteroglobin.
Peter
W
W
Institut für Molekularbiologie und Tumorforschung, Marburg, FRG.
Beato
M
M
Suske
G
G
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification; D004717:Endometrium / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D006023:Glycoproteins / Q000235:genetics*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D011247:Pregnancy; D011374:Progesterone / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014598:Uteroglobin / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D006023:Glycoproteins; D011994:Recombinant Proteins; D011374:Progesterone; D004247:DNA; D014598:Uteroglobin