Biochimica et Biophysica Acta, 1090 (199i) 293-298 © 1991 Elsevier Science Publishers B.V. All rights reserved 0167-4781/91/$03.50 293 BBAEXP 92302 Differential expression in Escherichia coli of the a and/3 forms of heparin-binding acidic fibroblast growth factor-l: potential role of RNA secondary structure Reza Forough I, Kurt Engleka l, John A. Thompson 2, Anthony Jackson i, Toru Imamura 3 and Thomas Maciag 1 / Laboratory of Mok'cular Bioh~gy, Jerome H. Holland Lahoratoo, for the Biomedical Sciences, American Red Cross, Rockrille, MD (U.S.A.), " Department of Biochemistly, Uniremity of Ahtbama at Birmingham, Birmingham, AL (U.S.A.) and "¢ Cell Science am/Technoh)gy Division, Fermentation Research Institute, Iharaki (Japan) (Received I April I()91) Key words: Fibroblast growth factor: Translation; RNA secondary structure Synthetic DNA fragments encoding the entire open-reading frame of human heparin-binding growth factor-I (HBGF-I/3) and its NH2-terminal truncated form (HBGF-Ia) were constructed. When both constructs were expressed in Escherichia coli under control of the trp-lac promoter, biologically active HBGF-Ia, but not HBGF-Ifl was produced in high yield. However. high level expression of HBGF-I/~ was obtained using the T7 polymerase expression vector. Computer analysis of HBGF-I/~ predicts the potential for the formation of exaggerated RNA secondary structure near the translation initiation codon and this could be implicated in contributing to the poor translation of HBGF-Ifl under the trp-lac promoter. Introduction Heparin-binding acidic fibroblast growth factor-I (HBGF-l) is a multipotent polypeptide growth factor characterized by its abilities to stimulate cell prolifera- tion and neurite extension in vitro and to induce neo- vascularization in vivo [1,2]. In addition, HBGF-I has been found associated with a number of pathophysio- logic conditions including arthritis and tumor angio- genesis [3,4]. Complementary DNA clones encoding human HBGF-l have been isolated and sequenced [5] and the translation of the full-length HBGF-1/3 open reading frame (ORF) in E. coli has proved to be difficult [6]. To study the significance of the 5' end of the HBGF-10RF with respect to its translational efficiency, we constructed a synthetic gene for the Abbreviations: HBGF-I, heparin-binding growth factor-!; ORF, open reading frame; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; IPTG, isopropylthiogalactoside; PCR, poly- merase chain reaction. Correspondence: T. Maciag, Laboratory of Molecular Biology, Jerome H. Holland Laboratory for the Biomedical Sciences, Ameri- can Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855, U.S.A. precursor (HBGF-1/3)and the processed form (HBGF- l a) of the growth factor and examined their expression in different prokaryotic expression vectors. Materials and Methods Construction of expression plasmids. Five synthetic DNA fragments encoding parts of the human HBGF-I ORF (Fig. IA) were assembled with oligonucleotides synthesized on an Applied Biosystem 380B DNA syn- thesizer by the phosphoromidite method and purified by polyacrylamide gel electrophoresis. Each fragment was independently cloned into EcoRl/Hindlll unique restriction sites of pDS25, a plasmid containing both the pBR322 and M13 phage origins of replication (Fig. I B). Briefly, mini-scale DNA prepared for each con- struct by the alkaline-lysis method was digested with appropriate restriction enzymes and subjected to 1% agarose (Bethesda Research Laboratories) gel elec- trophoresis. Appropriate DNA fragments were puri- fied from the gel by the phenol-freeze method [8] and were used for the assembly of the final constructs, HBGF-la and its precursor HBGF-I/3 in E. coli ex- pression vector pKK233-2 under control of the trp-lac promoter (Pharmacia Biotechnology) (Fig. 1C). The polymerase chain reaction (PCR) was used to add