Molecular gene cloning, expression, and characterization of bovine brain glutamate dehydrogenase.
A cDNA of bovine brain glutamate dehydrogenase (GDH) was isolated from a cDNA library by recombinant PCR. The isolated cDNA has an open-reading frame of 1677 nucleotides, which codes for 559 amino acids. The expression of the recombinant bovine brain GDH enzyme was achieved in E. coli. BL21 (DE3) by using the pET-15b expression vector containing a T7 promoter. The recombinant GDH protein was also purified and characterized. The amino acid sequence was found 90% homologous to the human GDH. The molecular mass of the expressed GDH enzyme was estimated as 50 kDa by SDS-PAGE and Western blot using monoclonal antibodies against bovine brain GDH. The kinetic parameters of the expressed recombinant GDH enzymes were quite similar to those of the purified bovine brain GDH. The Km and Vmax values for NAD+ were 0.1 mM and 1.08 micromol/min/mg, respectively. The catalytic activities of the recombinant GDH enzymes were inhibited by ATP in a concentration-dependent manner over the range of 10 - 100 microM, whereas, ADP increased the enzyme activity up to 2.3-fold. These results indicate that the recombinant-expressed bovine brain GDH that is produced has biochemical properties that are very similar to those of the purified GDH enzyme.
Kim
Dae Won
DW
Department of Genetic Engineering, Division of Life Sciences, Hallym University, Chunchon 200-702, Korea.
Eum
Won Sik
WS
Jang
Sang Ho
SH
Yoon
Chang Sik
CS
Kim
Young Hoon
YH
Choi
Soo Hyun
SH
Choi
Hee Soon
HS
Kim
So Young
SY
Kwon
Hyeok Yil
HY
Kang
Jung Hoon
JH
Kwon
Oh-Shin
OS
Cho
Sung-Woo
SW
Park
Jinseu
J
Choi
Soo Young
SY
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005969:Glutamate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D005969:Glutamate Dehydrogenase