Premium
Molecular cloning of a cDNA for a small GTP binding protein, BRho, from the embryo of Bombyx mori and its characterization after expression and purification
Author(s) -
Uno Tomohide,
Nakasuji Ai,
Hara Wajiro,
Aizono Yasuo
Publication year - 2000
Publication title -
archives of insect biochemistry and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.576
H-Index - 66
eISSN - 1520-6327
pISSN - 0739-4462
DOI - 10.1002/(sici)1520-6327(200004)43:4<165::aid-arch2>3.0.co;2-c
Subject(s) - gtpgammas , gtp' , biology , biochemistry , complementary dna , fusion protein , gtp binding protein regulators , g protein , microbiology and biotechnology , guanosine diphosphate , enzyme , recombinant dna , guanosine triphosphate , gene , receptor
A cDNA clone encoding a small GTP binding protein (Brho) was isolated from an embryonic cDNA library of Bombyx mori that encoded a polypeptide with 202 amino acids sharing 60–80% similarity with the Rho1 family of GTP binding proteins. The effector site and one of the guanine nucleotide binding sites differed from other members of the Rho family. To characterize the biochemical properties of Brho, the clone was expressed in Escherichia coli as a glutathione S‐transferase (GST) fusion protein. The recombinant protein was purified to homogeneity with glutathione S‐Sepharose. The fusion protein bound [ 35 S] GTPγS and [ 3 H] GDP with association constants of 11 × 10 6 M –1 and 6.2 × 10 6 M –1 , respectively. The binding of [ 35 S] GTPγS was inhibited by GTP and GDP, but by no other nucleotides. The calculated GTP‐hydrolysis activity was 89.6 m mol/min/mol of Brho. Bound [ 35 S] GTPγS and [ 3 H] GDP were exchanged with GTPγS most efficiently in the presence of 6 mM MgCl 2 . These results suggest that Brho has a higher affinity for GTP than GDP, converts from the GTP‐bound state into the GDP‐bound state by intrinsic GTP hydrolytic activity, and returns to the GTP‐bound state with the exchange of GDP with GTP. Arch. Insect Biochem. Physiol. 43:165–172, 2000. © 2000 Wiley‐Liss, Inc.