Expression of biologically active recombinant keratinocyte growth factor. Structure/function analysis of amino-terminal truncation mutants.

D Ron,D.P Bottaro,P.W Finch,D Morris,J.S Rubin,S.A Aaronson

doi:10.1016/s0021-9258(18)53870-9

Abstract

Keratinocyte growth factor (KGF) is a newly identified member of the fibroblast growth factor (FGF) family (FGF-7). KGF is expressed by stromal fibroblasts and acts on epithelial cells in a paracrine mode. To facilitate structure/function studies, we utilized the T7 prokaryotic expression system to synthesize this growth factor. Recombinant KGF (rKGF) was mitogenic with a specific activity around 10-fold higher than native KGF. By in vitro mutagenesis, we generated a series of KGF mutants with sequential deletions of the amino-terminal domain, the most divergent region among different FGF members. Mutant proteins, produced in bacteria, were tested for their ability to bind heparin, bind and activate the KGF receptor, and induce DNA synthesis. Heparin binding properties were preserved with deletion of up to 28 amino-terminal residues of the mature KGF but lost by the deletion of an additional 10 residues. Biological activity of mutants with deletions of up to 10 residues was comparable to that of rKGF. However, deletion of 29 residues resulted in significantly reduced ability to stimulate KGF receptor tyrosine-kinase activity and DNA synthesis, although this mutant bound the receptor at high affinity. These characteristics of a partial agonist may be useful in the development of competitive antagonists of KGF action.

Highlights

RKGF (5 ng/ml) M426 Keratinocyte growth factor (KGF)”EGF (10 ng/ml) aFGF (10ng/ml) cpm * 170 f 64136 f 8 17,829 f 4,14073,659 f 8,330 13,090 f 4,50720,004 f 2,665 13,135 f 76520,550 f 4,128 47,645 f 13,217 immunoblot analysis using rabbit polyclonal antisera directed against a synthetic peptide derived from the KGF carboxylterminal sequence (Fig. 50).We investigated the abilities of K10, K11, and K12 mutants todisplace radiolabeled KGF from its receptor, trigger receptor-kinaseactivity, and stimulate Balb/MK DNA synthesis
The sequence corresponding to KGFresidues Ala31 to Thrlg4was amplified by PCR
When the concentration dependence of recombinant KGF (rKGF) mitogenic activity on Balb/MKcells was compared withthat of the native protein purified from M426 fibroblasts, we found that the dose required for half-maximal stimulation by rKGF was 1 ng/ml

Summary

Expression of Biologically Active Recombinant Keratinocyte Growth Factor

Biological activity of the polymerase chainreaction(PCR) (5, 6),as described under mutants with deletions of up to 10 residues was com- “Results.” The recombinant plasmid, propagated in HBlOclells, was parable to that of rKGF. The abbreviations used are:KGF,keratinocyte growthfactor; rKGF, recombinant KGF; FGF, fibroblagsrot wth factor; aFGF,acidic FGF;bFGF, basic FGF;PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis. Balb/MK cells, plated in 96-well microtiter plates, were serum starved for 48 h, treated with growth factor, and processed as described previously (2) These cells, and theirgrowth was dramatically inhibitedeven by basal levels of KGF expression in thaebsence of isopropyl. 1-thio-/3;-D-galactopyranosidienduction.Wenext analyzed BL21(DE3) plysE cells, whichexpress a T7 RNApolymerase-

RESULTS

We demonstrate that recombinant expression of KGF in

Methods

Accumulating evidence indicates that a number of growth