Expresión of human Epidermal Growth Factor (hEGF) in tobacco chloroplasts

S. WIRTH, M.E. SEGRETIN, A. MENTABERRY, F.F. BRAVO-ALMONACID

Boca Chica, República Dominicana

Congreso; Vº Encuentro Latinoamericano y del Caribe de Biotecnología Vegetal; 2004

Redbio

High expression yields and low purification costs of a biologically active product are the main objectives for the design of a biopharmaceutical production system. In a recent work we demonstrated that biologically active hEGF can be expressed in transgenic and in viral vector infected tobacco plants (Wirth, et al, 2004).  Although hEGF expression levels reached in transgenic plants (0.11% of total soluble proteins) have still been evaluated for its accuracy for commercial production we decided to explore alternative systems to increase hEGF yields.  As chloroplast expression has demonstrated to be a feasible option for many proteins, we cloned hEGF gene in two different vectors we designed for tobacco chloroplast transformation. In the first vector, pBSWEGF, the hEGF gene is cloned with a consensus Shine Dalgarno sequence downstream of the aadA gene that confers spectinomycin resistance. The whole bicistron is under the transcriptional control of the chloroplast prrn promoter. In the second vector, pBSWUTREGF, the 5’ UTR region of chloroplast psbA gene is inserted between aadA and hEGF gene. N. tabacum Petit Havana plants leaves were transformed by bombardment with DNA coated gold particles using a Bio-Rad PDS-1000/He biolistic device and subjected to at least three rounds of regeneration in selective media. Homoplastic state of the regenerants and the insertion of the construction in the expected position in chloroplast genome were evaluated by PCR and southern blot analysis. Five transformed plants with each construction were selected at random for further analysis.  Northern blot analysis using specific probes demonstrated the high rate of transcription and showed the expected RNA sizes predicted by the position of the promoters inserted in the construction and the read-trough transcription by the chloroplast promoters according to the insertion site. Wirth S. et. al. 2004. Expression of active human epidermal growth factor (hEGF) in tobacco plants by integrative and non-integrative systems. Mol Breed. 13: 23