Expression of Recombinant Bovine γB-, γC- and γD-Crystallins and Correlation with Native Proteins

Re, Hay; Andley, Usha P.; Jm, Petrash

doi:10.1006/exer.1994.1052

Cited by 22 publications

(27 citation statements)

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“…For expression studies, plasmids were introduced into E. coli strain JM101. Cultures (1.6 liters) were grown in baffled shaker flasks as described previously (30,31). Expression of crystallins was induced in logarithmic cultures by addition of isopropyl-1-thio-␤-D-galactopyranoside to a final concentration of 1 mM and cultures were grown for an additional 12-18 h. Cells from a typical culture (A 600 ϳ 6) were collected by centrifugation (15,000 ϫ g, 15 min, 4°C) and were extracted by treatment with DNase (Sigma) and lysozyme essentially as described (32).…”

Section: Methodsmentioning

confidence: 99%

“…FPLC Gel Permeation Chromatography-In some cases, recombinant crystallins were purified by FPLC chromatography on Hi-Load 16/60 Superdex 200 (Pharmacia Biotech Inc.) gel as described previously (31). The gel filtration buffer contained 50 mM Tris-HCl, pH 7.6, 50 mM NaCl, and 1 mM EDTA.…”

Section: Methodsmentioning

confidence: 99%

“…Fluorescence spectra were measured with a Perkin-Elmer MPF-66 spectrofluorometer. Circular dichroism spectra were measured with a Jasco 600 spectropolarimeter, as described previously (31). Far-UV CD measurements were made between 200 -250 nm with 1-mm pathlength cells, and near-UV CD spectra were measured between 250 and 350 nm with 1-cm pathlength cells.…”

Section: Methodsmentioning

confidence: 99%

“…Immunological Methods-Antibodies against purified bovine lens ␣-crystallin were prepared in rabbits essentially as described previously for other antigens (31). For immunoblot analysis, proteins resolved by SDS-PAGE were electroblotted to nitrocellulose or Immobilon membranes (Millipore Corp., Bedford, MA) and were probed with antisera (diluted 1000-fold) under the conditions used previously (37).…”

Section: Methodsmentioning

confidence: 99%

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Cloning, Expression, and Chaperone-like Activity of Human αA-Crystallin

Andley

Mathur

Griest

et al. 1996

Journal of Biological Chemistry

Self Cite

152

136

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One of the major protein components of the ocular lens, ␣-crystallin, is composed of ␣A and ␣B chain subunits that have structural homology to the family of mammalian small heat shock proteins. Like other small heat shock proteins, ␣-crystallin subunits associate to form large oligomeric aggregates that express chaperone-like activity, as defined by the ability to suppress nonspecific aggregation of proteins destabilized by treatment with a variety of denaturants including heat, UV irradiation, and chemical modification. It has been proposed that age-related loss of sequences at the C terminus of the ␣A chain subunit may be a factor in the pathogenesis of cataract due to diminished capacity of the truncated crystallin to protect against nonspecific aggregation of lens proteins. To evaluate the functional consequences of ␣-crystallin modification, two mutant forms of ␣A subunits were prepared by site-directed mutagenesis. Like wild type (WT), aggregates of ϳ540 kDa were formed from a tryptophan-free ␣A mutant (W9F). When added in stoichiometric amounts, both WT and W9F subunits completely suppressed the heat-induced aggregation of aldose reductase. In contrast, subunits encoded by a truncation mutant in which the Cterminal 17 residues were deleted (R157STOP), despite having spectroscopic properties similar to WT, formed much larger aggregates with a marked reduction in chaperone-like activity. Similar results were observed when the chaperone-like activity was assessed through inhibition of ␥-crystallin aggregation induced by singlet oxygen. These results demonstrate that the structurally conservative substitution of Phe for Trp-9 has a negligible effect on the functional interaction of ␣A subunits, and that deletion of C-terminal sequences from the ␣A subunit results in substantial loss of chaperone-like activity, despite overall preservation of secondary structure.The major components of the mammalian lens fiber cells are the ␣-, ␤-, and ␥-crystallins, which constitute an estimated 35% wet weight of the lens. The crystallins contribute to the transparency and refractive power of the lens by short range interactions among themselves and cytoskeletal elements in a highly concentrated matrix (1-3). ␣-Crystallin is one of the most abundant of the crystallins in mature lens fiber cells. It is a M r ϳ0.6 -1.0 ϫ 10 6 complex composed of two structurally related subunits, designated ␣A and ␣B, which are encoded by genes localized to chromosomes 21 and 11, respectively (4, 5).

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Cloning, Expression, and Chaperone-like Activity of Human αA-Crystallin

Andley

Mathur

Griest

et al. 1996

Journal of Biological Chemistry

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152

136

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“…13) by using the method of Peitsch (14)(15)(16). The human ␥D protein sequence shares 87% identity with bovine ␥D and 78% identity with bovine ␥B in the primary structure (17,18). The comparative modeling was carried out in the automated internet server Swiss Model (www.expasy.ch), and the structures were displayed by using the personal computer version of the SWISS-PDB viewer.…”

Section: Se-hplcmentioning

confidence: 99%

Molecular basis of a progressive juvenile-onset hereditary cataract

Pande

Asherie

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

130

184

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We have expressed recombinant wild-type human ␥D crystallin (HGD) and its Arg-14 to Cys mutant (R14C) in Escherichia coli and show that R14C forms disulfide-linked oligomers, which markedly raise the phase separation temperature of the protein solution. Eventually, R14C precipitates. In contrast, HGD slowly forms only disulfide-linked dimers and no oligomers. These data strongly suggest that the observed cataract is triggered by the thiolmediated aggregation of R14C. The aggregation profiles of HGD and R14C are consistent with our homology modeling studies that reveal that R14C contains two exposed cysteine residues, whereas HGD has only one. Our CD, fluorescence, and differential scanning calorimetric studies show that HGD and R14C have nearly identical secondary and tertiary structures and stabilities. Thus, contrary to current views, unfolding or destabilization of the protein is not necessary for cataractogenesis.I n the hereditary, juvenile-onset cataract described by Stefan et al. (1), the lens, which is clear at birth, develops punctate opacities progressively, such that by two years of age the cataract is readily detectable, and matures by early childhood or adolescence. The punctate opacities seen in this cataract are in the nucleus and inner cortex, regions of the lens that are enriched in the ␥-crystallins. In the human lens, only two members of the ␥-crystallin family, ␥C and ␥D, are expressed in appreciable amounts, and only ␥D crystallin continues to be expressed until late childhood (2, 3). In affected individuals, a single point mutation has been identified in the ␥D crystallin gene that corresponds to the substitution of Arg-14 by a Cys. The identification of this mutation and the parallel between the time course of the pathology and the physiological expression of human ␥D crystallin strongly implicate the Arg-14 3 Cys mutant of ␥D in the development of this cataract. However, the molecular mechanism invoked to explain the observed opacity has been speculative (1).In the past, it has not been possible to conduct detailed studies on human ␥D crystallin because of the difficulty of obtaining sufficient quantities of pure protein from young, normal human lenses (4). Therefore, to characterize the normal protein thoroughly and investigate the mechanism by which the Arg-14 3 Cys mutation in ␥D could lead to cataract, we cloned and expressed human ␥D crystallin and its Arg-14 3 Cys mutant in Escherichia coli. Both the wild-type recombinant ␥D crystallin (HGD) and its Arg-14 3 Cys mutant (R14C) folded efficiently in E. coli and accumulated as soluble proteins. We isolated and purified the HGD and R14C proteins and determined their solution properties. Our results suggest that the disulfidecrosslinked oligomerization of R14C is responsible for the observed cataract. Furthermore, such oligomerization occurs without significant change in protein structure, conformation, and stability. Materials and MethodsCloning, Expression, and Isolation of Proteins. The human ␥D crystallin coding sequence was amplif...

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Cataract as a Protein‐Aggregation Disease

Wang

King

2010

Protein Misfolding Diseases

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Expression of Recombinant Bovine γB-, γC- and γD-Crystallins and Correlation with Native Proteins

Cited by 22 publications

References 0 publications

Cloning, Expression, and Chaperone-like Activity of Human αA-Crystallin

Cloning, Expression, and Chaperone-like Activity of Human αA-Crystallin

Molecular basis of a progressive juvenile-onset hereditary cataract

Cataract as a Protein‐Aggregation Disease

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