PARENT SESSION
Follicular Development

CLONING AND EXPRESSION OF BIOACTIVE RABBIT EPIDERMAL GROWTH FACTOR.

Floyd, Anthony¹, Lee, Vaughan ¹, Clarkson, Alison¹, ¹

ABSTRACT- Epidermal growth factor (EGF) is synthesized in many mammalian ovaries and has been implicated as a modulator of follicular growth and function. Investigation of the role of EGF in folliculogenesis has necessitated the production of large quantities of bioactive EGF for a variety of species. Characterization, cloning, expression, and testing of bioactivity for rabbit EGF (rbEGF) are described. A cDNA for rbEGF was generated by RT-PCR from RNA extracted from rabbit salivary glands and sequenced. The predicted amino acid sequence was compared to human EGF (81.1% identity), mouse EGF (71.7% identity), and pig EGF (88.7% identity). As in other forms of EGF studied, rbEGF shows conservation of Gly¹⁸, Tyr³⁷, Gly³⁹, Arg⁴¹, all six cysteine residues, and Leu⁴⁷ which are known to be essential for bioactivity. The cDNA encoding the 53 amino acid form of rbEGF was subcloned into a yeast (Pichia pastoris) expression system containing an -secretory sequence that facilitates secretion into the media and a C-terminal polyhistidine tag that forms a metal-binding site for affinity purification. Advantages of using a yeast expression system include enhanced expression, secretion into media which facilitates recovery and characteristics inherent to eukaryotic systems including proper protein processing, folding, and post-translational modification. The recombinant protein was purified from media using Ni-NTA columns, assayed, and characterized by Western immunoblotting. Bioactivity was evaluated using mouse 3T3 fibroblast cell proliferation assays, with potency compared to previously tested, commercially available mouse EGF. ED₅₀ data for rbEGF and mouse EGF were similar, ranging from 0.1 to 0.5 ng/ml. Further testing showed stimulation of Cx43 expression in rabbit granulosa cells. This stimulation was optimized at doses of 10 to 50 ng/ml, similar to results for mouse EGF. These results support the utility of this yeast expression system in producing substantial quantities of bioactive rbEGF protein to be used in reproductive as well as other biological studies.

KEY WORDS: EGF, yeast expression systems