Doctoral Dissertations

Date of Award

8-1990

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Audrey Stevens

Committee Members

Salil K. Niyogi, Robert K. Fujimura, Lee R. Shugart, Fred C. Hartman

Abstract

The gene for human epidermal growth factor was synthesized and placed behind the gene coding for the signal sequence of E. coli alkaline phosphatase. The chimeric gene was then placed in an expression vector behind the tac promoter and under the control of the lac repressor/operator and transformed into E. coli JM107, This construct allows for the expression of human epidermal growth factor and secretion of the protein into the periplasmic space of E. coli. Protein of correct length, and properly folded was isolated and found to be fully active in several biological assays: binding to the EGF receptor, stimulation of the receptor's tyrosine kinase, and stimulation of thymidine uptake in tissue culture cells. Site-directed mutagenesis was carried out at two conserved sites within the EGF molecule. The proline residue at position 7 was changed to a threonine and was found to have about 50% of the wild type activity in receptor binding and in tyrosine kinase stimulation. The leucine residue at position 47 was changed to an isoleucine, histidine, proline, alanine, glycine, aspartic acid, and arginine. The activities for these mutants in the receptor binding assays ranged from 17% to .06% of wild type with the order from best to worst as listed above. Results from the tyrosine kinase stimulation were similar to the binding results. Stimulation of thymidine uptake ranged from 44% to 0.4% of wild type, in approximately the same order. Three of the leucine mutants, isoleucine, alanine, and glycine, were examined for their structural integrity using nuclear magnetic resonance (NMR) spectroscopy. One dimensional proton NMR showed little structural perturbations in these mutants. The EGF receptor's tyrosine kinase activity was investigated more fully. Several tyrosine-containing synthetic polymers were tested for their ability to act as substrates for the EGF receptor tyrosine kinase. Polymers poor in tyrosine were found to be good substrates, while polymers rich in tyrosine were found to inhibit the phosphorylation of the tyrosine poor polymers. This inhibition appears to be of a mixed type. Tyrosine-rich polymers also inhibited receptor autophosphorylation. Several mutants of EGF were found to have a lower Vmax in the tyrosine kinase phosphorylation assay. These mutants were further examined and were shown to competitively inhibit the wild type protein in its ability to stimulate the receptor's tyrosine kinase activity.

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