Glutamic acid and alanine spacer is not necessary for removal of MFa-1 signal sequence fused to the human growth hormone produced from Pichia pastoris Lily Eurwilaichitr 1, *, Sittiruk Roytrakul 1 , Chittiwat Suprasongsin 2 , Pennapa Manitchotpisit 3 and Sakol Panyim 4 1 BIOTEC Training Center for Genetic Engineering and Biotechnology, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakhonpathom 73170 Thailand 2 Ramathibodi Hospital, Mahidol University, Rama 6 Road, Bangkok, Thailand 3 Department of Microbiology, Rangsit University, Bangkok, Thailand 4 Institute of Molecular Biology and Genetics, Mahidol University, Salaya campus, Nakhonpathom 73170, Thailand *Author for correspondence: Tel.: +66-2 441 9003-7, Fax: +66-2 441 9906, E-mail: lily@biotec.or.th Received 6 August 2001; accepted 21 March 2002 Keywords: a-Mating factor, glu-ala repeats, human growth hormone, PCR and Pichia pastoris Summary Human growth hormone (hGH) cDNA was synthesised using codons preferred by Escherichia coli, except for the first 20 amino acids, which were changed to that preferred by Saccharomyces cerevisiae and Pichia pastoris. Polymerase chain reaction (PCR) overlapping approach was employed to create synthetic hGH without glutamic acid-alanine (glu-ala), or with one and two glu-ala spacers (hGH, hGH1 and hGH2, respectively). The necessity of a glu-ala spacer in the cleavage of S. cerevisiae alpha mating factor-1 (MFa-1) secretion signal from the synthetic hGH was also investigated. Three types of hGH constructs were integrated into P. pastoris genome, the zeocin- resistant transformants were selected and expression of hGH was determined. A 22-kDa band of secreted hGH was further determined by N-terminal peptide sequencing. The result suggested that the removal of glu-ala from the hGH1 and hGH2 was not efficient and only the hGH construct showed the complete cleavage of the signal sequence, giving a similar N-terminus as the mature hGH. hGH expression was optimized to increase the yield of the protein from the hGH construct (no glu-ala) to 190 mg/l from a 10-ml induction medium. Introduction Human growth hormone (hGH) has been used in treatments of dwarfism, bone fractures and bleeding ulcers. The hormone molecule consists of 191 amino acid residues which folds into a four-helix bundle structure with two disulphide bridges (De Vos et al. 1992). Since hGH is a non-glycosylated protein, prok- aryotic expression systems such as Escherichia coli have been preferred in the production of recombinant hGH (Goeddel et al. 1979; Chang et al. 1987; Kato et al. 1987). However, it is reported that desired products expressed in E. coli are initiated with formylmethionine (fMet) which in many cases is not efficiently removed. It has been suggested that the fMet at the N-terminus of methionyl hGH may play a role in antibody formation in patients treated with the hormone (Glasbrenner 1986). Among eukaryotic expressions systems, Pichia pasto- ris, therefore, becomes an alternative host. It is a methylotrophic yeast which grows on methanol as a sole carbon source. Methanol oxidation is catalysed by an enzyme, alcohol oxidase, which may produce up to 35% of total protein in cells. The highly inducible and tightly regulated alcohol oxidase 1 gene promoter (AOX1) has been used to construct an expression vector (Sreekrishna et al. 1987). Pichia pastoris, therefore, provides extremely high yields of both intracellular protein and secreted protein into an almost protein-free medium by the use of a S. cerevisiae a-factor leader sequence. The cleavage sites for removal of the a-factor leader sequence provided in the P. pastoris vector is a Kex2 recognition site (lysine-arginine: lys-arg) which is followed by two glutamic acid-alanine (glu-ala) repeats. It is not clear at present if the two glu-ala repeats are essential for cleavage of the a-factor-protein in P. pastoris. In S. cerevisiae, it has been noted that the glu-ala repeats are not necessary for cleavage by Kex2, except if the first amino acid after Lys-Arg belongs to the groups of aromatic rings, small amino acids and histidine (Brake et al. 1984). The first amino acid of hGH is a phenylalanine, an aromatic amino acid. This might cause the tolerant in Kex2 cleavage, therefore Glu-Ala repeats might be necessary. In the current work, the hGH gene was synthe- sized using primer extension and PCR amplification World Journal of Microbiology & Biotechnology 18: 493–498, 2002. 493 Ó 2002 Kluwer Academic Publishers. Printed in the Netherlands.