March 2012
Volume 53, Issue 14
ARVO Annual Meeting Abstract  |   March 2012
High Efficiency Transduction Of α-crystallin Into Mammalian Cells
Author Affiliations & Notes
  • Niklaus H. Mueller
    Ophthalmology, Univ of Colorado School of Medicine, Aurora, Colorado
  • Spencer LaFasto
    Ophthalmology, Univ of Colorado School of Medicine, Aurora, Colorado
  • David Ammar
    Ophthalmology, Univ of Colorado School of Medicine, Aurora, Colorado
  • J. Mark Petrash
    Ophthalmology, Univ of Colorado School of Medicine, Aurora, Colorado
  • Footnotes
    Commercial Relationships  Niklaus H. Mueller, None; Spencer LaFasto, None; David Ammar, None; J. Mark Petrash, None
  • Footnotes
    Support  5RC1EY020361
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 3669. doi:
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      Niklaus H. Mueller, Spencer LaFasto, David Ammar, J. Mark Petrash; High Efficiency Transduction Of α-crystallin Into Mammalian Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3669.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : The loss of native α-crystallins is thought to be a risk factor for formation of protein aggregates, loss of lens transparency, and nuclear cataract in humans. The α-crystallins have been shown to prevent protein aggregation in vitro. We propose that replenishing intracellular α-crystallin by providing therapeutic levels of recombinant protein to lens cells may delay or prevent cataract formation. The purpose of the current study was to investigate the ability of a novel cell penetration peptide to enhance transduction of therapeutic crystallins into lens cells.

Methods: : Recombinant human αA- and αB-crystallins were modified by addition of a novel protein transduction peptide designed with reference to the structure of the herpes simplex virus gC gene product (HSV-1 gC peptide). Fusion proteins produced by placing the HSV-1 gC peptide at either the N- or C-terminus were over-expressed in E. coli cultures and were purified to apparent homogeneity. These proteins were subjected to size exclusion chromatography to characterize high molecular weight complexes (HMWC). Chaperone-like activity (CLA) of α-crystallins was evaluated by measuring the suppression of chemically-induced aggregation of substrate proteins. α-crystallins were fluorescently-labeled using Alexa-based dyes and were incubated with human lens epithelial cells (HLE B3) and human embryonic kidney cells (HEK293). Uptake of α-crystallins was monitored by fluorescent microscopy for 72 hrs.

Results: : We examined the effects of a peptide predicted from the HSV-1 gC gene (gC) on the structure, CLA, and cell transduction properties of human α-crystallins. Unlike the case with C-terminal fusion of the peptide, N-terminal tagged α-crystallin subunits expressed in E. coli were in the soluble fractions of host cell lysates. These modified α-crystallin subunits formed HMWC of ~650 kDa that were slightly larger than wild-type α-crystallins based on size exclusion chromatography. CLA of these proteins were similar to wild-type α-crystallins in their ability to suppress chemically-induced aggregation of substrate proteins for at least an hour. Also, modified α-crystallins underwent subunit exchange with wild type α-crystallin over a 24 hr period. When wild-type α-crystallin proteins were incubated with cultured cells, only limited protein uptake occurred. In contrast, protein uptake increased extensively when gC tagged α-crystallins were analyzed.

Conclusions: : gC tagged α-crystallin proteins, like wild-type α-crystallins, form HMWC, inhibit protein aggregation in vitro, and undergo subunit exchange. Addition of the gC fusion peptide conferred a substantially higher level of penetration into mammalian cells in a tissue culture setting.

Keywords: protein purification and characterization • chaperones • cataract 

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