2013 mb

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Mol Biotechnol (2013) 55:150–158 DOI 10.1007/s12033-013-9667-3

RESEARCH

Construction of an Expression Vector for Production and Purification of Human Somatostatin in Escherichia coli Sergi Maicas • Ismaı¨l Moukadiri • Almudena Nieto • Eulogio Valentı´n

Published online: 3 May 2013 Ó Springer Science+Business Media New York 2013

Abstract Somatostatin/growth hormone-inhibiting hormone is the peptide that inhibits secretion of somatotropin/ growth hormone. Solid-phase synthesis methods are being currently used to produce somatostatin. Recombinant peptide synthesis is widely described for the production of small proteins and peptides; however, the production at industrial scale of peptides for biopharmaceutical applications is limited for economic reasons. Here, we propose the use of a new pGB-SMT plasmid to produce Somatostatin, as a C-terminal fusion protein with a Kluyveromyces lactis b-galactosidase fragment. To facilitate removal of that fragment by CNBr cleavage, a methionine residue was introduced at the N-terminal of the hormone peptide. The use of this construction enables an IPTG-free expression system. The suitability of this procedure has been assessed in a 15 l scale-up experiment yielding almost 300 mg, with purity [99 % and it is being implemented for commercial scale. The plasmid pGB-SMT here described is an alternative option for a cheap and high expression of other short peptide hormones. Keywords Escherichia coli Expression FPLC HPLC Human somatostatin Ion exchange purification

S. Maicas (&) A. Nieto E. Valentı´n Departament de Microbiologia i Ecologia, Universitat de Vale`ncia, Dr. Moliner, 50, 46100 Burjassot, Spain e-mail: sergi.maicas@uv.es I. Moukadiri Laboratorio de Gene´tica Molecular, Centro de Investigacio´n ‘‘Prı´ncipe Felipe’’, Camino de las Moreras s/n, 46013 Valencia, Spain

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Introduction Somatostatin/growth hormone-inhibiting hormone (GHIH) was first discovered in hypothalamic extracts and identified as a hormone that inhibited the secretion of somatotropin/ growth hormone (GH) [1]. Although first described in the hypothalamus, Somatostatin-producing cells occur in many organs, including the central and peripheral nervous system, pancreas, gut, thyroid, adrenals, spleen, liver, kidneys and prostate. Furthermore, somatostatin is produced by inflammatory and immune cells [1]. It is synthesized as a precursor molecule, preprosomatostatin, which after processing generates two bioactive forms, somatostatin-14 and somatostatin-28. The two cysteine residues in the 14 amino acid peptide (NH2–A-G-C-K-N-F-F-W-K-T-F-T-S-C–COOH) allow the formation of an internal disulphide bond [2]. Somatostatin is produced in different quantities by different cells and, at least in the rat, the gut accounts for 65 % of total body somatostatin-like immunoreactivity, the brain for 25 %, the pancreas for 5 % and the remaining organs for 5 % [3]. Nutrients (glucose, amino acids and lipids), neurotransmitters, neuropeptides (glucagon, GH releasing and bombesin), hormones (insulin and glucocorticoids) and cytokines (interleukin-1, interleukin-6, transforming growth factor-b, tumour necrosis factor-a, insulinlike growth factor, leptin or interferon-c), and several intracellular mediators including cyclic AMP, cyclic GMP, Ca2? and nitric oxide, all influence the expression and/or secretion of somatostatin [4, 5]. Somatostatin acts as a neurotransmitter and as an autocrine, paracrine or endocrine regulator. It controls many physiological functions including modulation of neurotransmission, cell secretion and proliferation, smooth muscle cell contractility, intestinal motility, absorption of nutrients and immune cell functions [5].


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