December 2002
Volume 43, Issue 13
Free
ARVO Annual Meeting Abstract  |   December 2002
Expression of Recombinant Transforming Growth Factor Beta Inducible Protein in Insect Cells
Author Affiliations & Notes
  • BS McKay
    Duke University Durham NC
    Ophthalmology & Cell Biology
  • W Bao
    Ophthalmology
    Duke University Durham NC
  • GK Klintworth
    Ophthalmology and Pathology
    Duke University Durham NC
  • Footnotes
    Commercial Relationships   B.S. McKay, None; W. Bao, None; G.K. Klintworth, None. Grant Identification: NEI Grant #P30 EY05722. RPB
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 1735. doi:
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      BS McKay, W Bao, GK Klintworth; Expression of Recombinant Transforming Growth Factor Beta Inducible Protein in Insect Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1735.

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

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Abstract

Abstract: : Purpose: Transforming growth factor beta induced protein (tgfbip) is a ubiquitous protein of unknown function present in blood as well as many tissues including the cornea. Mutations in the TGFBI (BIGH3) gene encoding for this protein are responsible for several distinct inherited corneal disorders. Here we attempt to express and purify large quantities of the wild-type and mutant tgfbip from a eukaryotic expression system capable of secreting the recombinant protein with the correct disulfide bonding patterns and native folding. Method: We received a gift of a plasmid containing TGFBI cDNA from Bristol Myers Squib. Site-directed mutagenesis was performed to create point mutations. Wild-type and mutated TGFBI with an attached carboxyl Hisx6 purification tag were cloned into the baculovirus shuttle vector BacPak8. Recombinant baculovirus was obtained by in vivo recombination in SF9 (Spodoptera frugipoda) cells. Protein expression was performed in HighFive (Trichoplusia ni) insect cells grown in ultralow protein, serum-free medium. The recombinant proteins were partially purified using a nickel chelation column and protein expression was verified by western blot analysis using a peptide-directed antibody developed in rabbits. Results: Both the wild-type and the mutant recombinant protein were expressed by HighFive cells. Much of the protein was either cell or substrate associated and it could be detected using the peptide directed antibody, and no bands were evident in cultures infected with non-recombinant virus (negative control). The protein expressed in insect cells exhibits an apparent molecular weight of 67kD, similar to that from human corneas. Conclusion: Our data illustrate the use of a eukaryotic expression system to express and purify tgfbip in the absence of serum contaminants. The recombinant proteins exhibit a size similar to that observed in humans and offer the use of a eukaryotic expression system to test the structure and function of the wild-type and mutant tgfbip. Such work should lead to an explanation of the corneal disorders characterized by an abnormal accumulation of tgfbip within the cornea.

Keywords: 370 cornea: basic science • 385 degenerations/dystrophies • 526 protein purification and characterization 
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