Expression of recombinant bovine γB-, γC- and γD-crystallins and correlation with native proteins

Regine E. Hay, Usha P. Andley, J. Mark Petrash

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22 Scopus citations

Abstract

Despite the use of bovine γ-crystallins in numerous biophysical and chemical studies characterization of these proteins at the molecular level is incomplete. Bovine lenses have at least sixγ-crystallin protein fractions currently assigned as γs/I, γA/IVb, γB/II, γC/III, γD/IIIa and γE/IVA. A lack of primary sequence data for corresponding γ-crystallin genes and proteins, however. has made these assignments tenuous. To clarify these assignments, we have over-expressed recombinant bovine γ-crystallin proteins in Escherichia coli using complementary DNAs corresponding to γB-, γC-, and γD-crystallin genes. The recombinant crystallins were characterized by chromatographic and spectroscopic comparisons with native bovine crystallin fractions γII, γIIIa and γIIIb. The elution of recombinant γB and native γII proteins was identical on cation-exchange chromatography as expected; however, recombinant γC coeluted with γIIIa and recombinant γD co-eluted with γIIIb. Sequential Edman degradation through the first 29 residues of the native γIIIa and γIIIb polypeptides confirmed the colinearity of their sequences with those predicted from the γC-, and γD-crystallin genes, respectively. Absorption and UV circular dichroism (CD) spectra of the recombinant proteins were almost identical to those of their native counterparts, indicating that the secondary and tertiary structures of the recombinant proteins were characteristic for γ-crystallins. Based on these data the bovine γ-crystallins proteins and genes are correlated as follows: II/γB, IIIa/γC and IIIb/γD. The assignment of γIIIb (previously characterized as having a low critical temperature for phase separation) with γD rather than γC proves an exception to the hypothesis that the γABC-crystallin group is more resistant to phase separation than the γDEF group. These corrected assignments should provide a more substantial base for investigations of residues responsible for phase separation and other biophysical characteristics. Additionally, expression of recombinant γ-crystallins with structures similar to native proteins may prove to be useful in probing specific structure-function relationships of the γ-crystallins.

Original languageEnglish
Pages (from-to)573-584
Number of pages12
JournalExperimental eye research
Volume58
Issue number5
DOIs
StatePublished - May 1994

Keywords

  • Cataract
  • Lens crystallins
  • Polymerase chain reaction
  • Protein sequence
  • Recombinant protein
  • Spectroscopy
  • γ-crystallin cDNAs

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