Frank Giblin and His Team Purify a Protein Found in the Eye

"Our laboratory has found that chronic exposure of guinea pigs to UVA [ultraviolet A] light produces detrimental effects in the central region of the lens including increased light scattering, disulfide crosslinking of proteins and high molecular weight aggregate formation... We hypothesize that these effects are caused by absorption of UVA light in the guinea pig lens by NADPH [Nicotinamide adenine dinucleotide phosphate] bound to f-crystallin [a water-soluble structural protein found in the lens of the eye], leading to the generation of reactive oxygen species [small, highly reactive molecules that contain oxygen]…such as superoxide anion [O₂^-] and H₂O₂ [Hydrogen peroxide]. By absorbing UVA light in the lens, f-crystallin may act to protect the guinea pig retina…from UVA-induced damage. Indeed, retinas of guinea pigs that have been made aphakic [lacking a lens] have been reported to be damaged by…UVA light… Similar events may occur in the aging human lens where the binding of the UVA chromophore kynurenine [a metabolite of the amino acid tryptophan used in the production of niacin] to crystallins may lead to subsequent generation of damaging reactive oxygen species.

The purpose of the present study was to employ recombinant methods [processes in which a molecule of DNA is broken and then joined to a different DNA molecule] to produce substantial amounts of f-crystallin which could later be used for in vitro investigation [in an articial environment like a test tube, as opposed to in a live animal] of mechanisms of UVA-induced damage in the lens. f-Crystallin has been cloned previously from cDNA libraries [DNA synthesized from a messenger RNA in a reaction catalyzed by the enzyme reverse transcriptase] of guinea pig lens epithelial cells…guinea pig lenses…guinea pig and mouse liver…and human liver….  Other investigators have found that attempts to express recombinant guinea pig f-crystallin in Escherichia coli [a bacterium often used when producing proteins using recombinant DNA methods] resulted in low yields since the expressed protein deposited mainly into inclusion bodies [aggregates of proteins] …. In the current study, histagged [an amino acid sequence in proteins that consists of at least six copies of the amino acid histidine, and are often used when producing genetically modified proteins] recombinant mouse f-crystallin was overexpressed in E. coli in substantial amounts … and the protein was purified to homogeneity."