Crocodilian tau-crystallin: overexpression, purification, and characterization.
tau-Crystallin is a taxon-restricted crystallin found in eye lenses of reptiles and a few avian species but presumably absent in mammals. The level of tau-crystallin in the lens varies among different species. In the crocodile lens, it is the least abundant crystallin and is present in trace amounts. We present a method for cloning, overexpression, and purification of crocodilian tau-crystallin utilizing a combination of gel filtration and ion-exchange chromatography yielding an extremely purified protein. The protein gets profusely expressed resulting in a fairly high yield and exists as a monomeric entity of 47.5 kDa molecular mass. The recombinant tau-crystallin exists in a properly folded native state as probed by circular dichroism and fluorescence spectroscopy and exhibits enolase activity.
Mishra
Anurag Kumar
AK
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, India.
Chandrashekhar
Reena
R
Aggarwal
Ramesh K
RK
Sharma
Yogendra
Y
D000489:Alligators and Crocodiles; D000818:Animals; D001671:Biochemistry / Q000379:methods*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016622:Silver Staining; D013050:Spectrometry, Fluorescence; D038225:tau-Crystallins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification*
D038225:tau-Crystallins; D010751:Phosphopyruvate Hydratase