High level expression of rat gamma-D-crystallin in Escherichia coli.
Gamma-crystallins have been implicated in various kinds of cataracts. In order to facilitate studies elucidating the molecular mechanism of cataractogenesis, large quantities of rat recombinant gamma-D-crystallin were produced in E coli. A full length cDNA clone coding for gamma-D-crystallin was isolated from a rat lens lambda gt11 cDNA library using a synthetic oligonucleotide as a probe. The coding region of this cDNA was inserted into a cloning vector pKK233-2 under the control of the trc promoter. The resulting construct, pKKCR91, was transfected into E coli to produce rat gamma-D-crystallin in an amount of 10-15% of the total bacterial proteins. The crystallin produced was purified to an apparent homogeneity as judged by SDS gel electrophoresis. The sequence of the N-terminal 11 amino acids of the purified crystallin was determined, showing that it is completely identical to that predicted from the cDNA sequence. Measurements of the far-UV CD spectra also revealed that recombinant rat gamma-D-crystallin thus produced retains a native conformational structure.
Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan.
K
K
Ooki
N
N
Amuro
Y
Y
Shimizu
T
T
Okazaki
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003459:Crystallins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004269:DNA, Bacterial; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D017433:Protein Structure, Secondary; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D003459:Crystallins; D004269:DNA, Bacterial; D018076:DNA, Complementary; D011994:Recombinant Proteins