Biotechnology Letters 22: 109–114, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. 109 Biosynthesis and secretion of recombinant human growth hormone in Pichia pastoris L.L. Ecamilla-Treviño, J.M. Viader-Salvad´ o, H.A. Barrera-Saldaña & M. Guerrero-Olazar´ an 1,∗ Departamento de Bioqu´ ımica, Facultad de Medicina, Universidad Aut´ onoma de Nuevo Le´ on, Ave. Madero y Dr. Eduardo Aguirre Pequeño, Col. Mitras Centro, 64460 Monterrey, N.L., M´ exico 1 Present address: Departamento de Microbiolog´ ıa, Facultadde Medicina, U.A.N.L., Av. Madero y Dr. Eduardo Aguirre Pequeño, Col. Mitras Centro, 64460 Monterrey, N.L., M´ exico ∗ Author for correspondence (Fax: +528 329-4166; E-mail: mguerrer@ccr.dsi.uanl.mx) Received 27 August 1999; Revisions requested 30 September 1999; Revisions received 18 November 1999; Accepted 22 November 1999 Key words: alcohol oxidase, gene expression, human growth hormone, Pichia pastoris Abstract Mature human growth hormone (hGH) cDNA was cloned by homologous recombination into the yeast Pichia pastoris genome. The hGH gene expression was placed under the control of the methanol-inducible alcohol oxidase 1(AOX1) gene promoter and the Saccharomyces cerevisiae α-factor signal sequence to direct the secretion of recombinant human growth hormone (rhGH) into the growth medium. O 2 -limited induction of recombinant yeast strains in shake tubes with 3 ml of culture medium produced up to 11 mg rhGH l −1 , while high cell density cultures using a 2-l bioreactor produced about 49 mg rhGH l −1 achieving 40% of total protein of the culture medium supernatant. Introduction Pichia pastoris is a methylotrophic yeast that has been shown to be an outstanding host for high-level hetero- logous gene expression (Cregg et al. 1993, Romanos 1995, Sreekrishna et al. 1997). The success of the Pichia pastoris expression system is linked to the tightly-regulated alcohol oxidase (AOX1) promoter. The strong promoter coupled with the high cell density fermentations, using a simple methanol salt medium, allow high level productions of intracellular and extra- cellular recombinant proteins. Recombinant proteins are secreted into the culture medium with the particu- lar advantage that this yeast secretes only low levels of endogenous protein, thus secretion serves as first step in protein purification (Cregg et al. 1993, Romanos 1995, Sreekrishna et al. 1997, Higgins & Cregg 1998). Human growth hormone (hGH) is a 191-amino acid protein with a molecular weight of ∼22 kDa whose recombinant version is successfully used to treat hypopituitary dwarfism. In addition to treatment of children with growth hormone deficiency, admin- istration of hGH appears to be of considerable be- nefit to girls with Turner’s syndrome, children with chronic renal failure and adults with growth hormone deficiency or human immunodeficiency virus (HIV) syndrome. Likewise, its therapeutic use is being in- vestigated in others therapeutic applications, such as children with idiopathic short stature, maintaining health in the elderly and the critically ill (Tritos & Mantzoros 1998). Production of rhGH has been reported mostly from Escherichia coli (Goeddel et al. 1979, Gray et al. 1985, Becker et al. 1986, Kato et al. 1987, Hsiung et al. 1989). In this host, rhGH has been produced in an intracellular approach, secreted into E. coli peri- plasm or even secreted into culture medium using specialized secretion vectors. rhGH has been obtained also from Bacillus subtilis, Saccharomyces cerevisiae and Pseudomonas recombinant strains (Franchi et al. 1991, Tokunaga et al. 1985, Gray et al. 1984). As an example of application of the P. pastoris system, in the present work, a new approach for the production and secretion of mature and biolo-