Differential expression of human basic fibroblast growth factor in Escherichia coli: potential role of promoter Hasan Mirzahoseini*, Farideh Mehraein, Eskandar Omidinia and Mohamad R. Razavi Department of Biochemistry, Pasteur Institute of Iran, Pasteur St. No. 69, Tehran 13164, Iran *Author for correspondence: Tel.: +98-640-2770, Fax: +98-646-5132, E-mail: Mirzahoseini@institute.pasteur.ac.ir, Mirzahoseini@yahoo.com Received 21 October 2002; accepted 27 September 2003 Keywords: hbFGF, inclusion body, plasmid, promoter Summary Four different expression systems were developed for expression of the cDNA encoding human basic fibroblast growth factor (hbFGF), using Escherichia coli as host organism. The hbfgf structural gene was cloned into four expression vectors, pET-3a, pTrc99A, pPR37 and pKK223-3 differing only in their promoters, which were T7, trc, kPR and tac respectively. Expression of the gene was induced by adding 0.5 mM (final concentration) of isopropyl- b-D -thio-galactopyranoside (IPTG) for the vectors carrying T7, trc and tac promoters or by a temperature shift from 30 to 42 °C for the vector carrying kPR. The highest level of expression was observed in pET-1005 (a pET-3a derivative)/BL21 (DE3) system with 18.5 mg/l rhbFGF and the second high level expression was in pR37-1007 (pPR37 derivative) BL21 (DE3) system with 5 mg of rhbFGF/l. Since in the latter system a temperature shift was used for induction, 29% of the hbFGF was recovered as inclusion bodies in the insoluble cell fraction. The level of expression for the two other systems (pTrc-1006 and pKK-1008) was very low. Introduction Human basic fibroblast growth factor (hbFGF) or FGF-2 is a heparin-binding growth factor which has pleiotropic roles in many cell types and tissues; it is a motogenic, angiogenic and survival factor which is involved in cell migration, cell differentiation and in a variety of developmental processes (Okada-Ban et al. 2000). The hbFGF has attracted profound interest in the pharmaceutical industry due to several potential appli- cations, such as wound healing and redirection of blood vessels (Anspach et al. 1995). Economic processes for the production of recombinant pharmaceutical proteins require high specific product yields and the highest specific product concentrations are generally obtained by placing the structural gene downstream of strong promoters (Seeger et al. 1995). In this report we compare the performance of one temperature and three IPTG-inducible expression sys- tems in E. coli synthesizing hbFGF as a heterologous protein. In many cases, such as this study, over- expression of foreign proteins in hosts results in the formation of insoluble inclusion bodies. Deposition of the recombinant protein in inclusion bodies may or may not be advantageous, as will be discussed later. Materials and methods Preparation of cDNA hbFGF As we reported previously (Mirzahoseini et al. 2001), the cDNA of hbFGF was cloned into pUC18 plasmid (construct pUC-1003) and pET3a (construct pET-1005) expression vector. Subcloning of cDNA in pTrc99A The PCR was carried out using the pUC-1003, as template, a forward primer 34 bp 5¢AATT GAATTC TAA AGGA A AGCTTA T ATGCCCGCC 3¢ and a reverse primer 30 bp 5¢ TACTATTAGATCT- TGGCCATTAAAATCAGC 3¢ (made in the Institute of Bioorganic Chemistry, Moscow). The forward primer carries an EcoRI site, a termination codon (TAA), a ribosome binding site (rbs), a HindIII site and a start codon (ATG) in tandem, while in the reverse primer only BglII site was inserted. The pTrc99A statement vector (purchased from Pharmacia) carries a trc promoter upstream of the polycloning site (PCS). In PCS, an rbs sequence is followed by a NcoI site and a termination codon is located after EcoRI site. The PCR product was cut by World Journal of Microbiology & Biotechnology 20: 161–165, 2004. 161 Ó 2004 Kluwer Academic Publishers. Printed in the Netherlands.