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Distinct Human Prolactin (Hprl) and Growth Hormone (Hgh)

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Distinct Human Prolactin (Hprl) and Growth Hormone (Hgh) Available online at www.sciencedirect.com Journal of Biotechnology 133 (2008) 27–35 Distinct human prolactin (hPRL) and growth hormone (hGH) behavior under bacteriophage lambda PL promoter control: Temperature plays a major role in protein yields C.R.J. Soares ∗, E.K.M. Ueda, T.L. Oliveira, F.I.C. Gomide, S.R. Heller, P. Bartolini Biotechnology Department, IPEN-CNEN, Av. Lineu Prestes, 2242 Cidade Universit´aria, 05508-900 S˜ao Paulo, Brazil Received 6 February 2007; received in revised form 21 August 2007; accepted 24 August 2007 Abstract When producing recombinant protein for therapy, it is desirable not only to obtain substantial amounts of the protein, but also to make sure that potential contaminants such as inducing agents are not present in the final product. To prevent this, one can use expression systems in which the promoter (␭PL) is activated by a temperature shift that denatures a repressor (e.g., cIts). In this manner, hGH was successfully expressed and ␮ −1 −1 ◦ secreted in Escherichia coli periplasm, with specific yields well above 1 gml A600, after a temperature shift from 30 to 42 C. However, attempts ␮ −1 −1 to express a related hormone, human prolactin, employing the same protocol were unsuccessful, providing 0.03 gml A600 at the most. A process is described in which this labile protein is obtained from a cIts− strain under optimized temperature condition (37 ◦C). The highest periplasmic ± ␮ −1 −1 ∼ secretions of prolactin ever reported were thus obtained: 0.92 0.10 gml A600 at an optical density of 3A600 units in shake flask cultures and ∼ ␮ −1 −1 1 gml A600,atanODof35A600 units, via a rapid and flexible batch feed process in laboratory bioreactor. Purified hPRL was monomeric, correctly processed (Mr = 22,906), properly folded and bioactive (51.5 ± 24.1 IU mg−1). © 2007 Elsevier B.V. All rights reserved. Keywords: Human growth hormone; Human prolactin; Escherichia coli periplasm; Lambda PL promoter; Thermo-sensitive repressor; Fed batch culture 1. Introduction lation), the basic hormone is a single-chain polypeptide with a calculated molecular mass of 22,897.75 Da, which has been con- Prolactin (PRL), a protein hormone primarily produced by the firmed by relative molecular mass (Mr) determinations (Wu et anterior pituitary, has various physiological functions in mam- al., 2003; Soares et al., 2006). It consists of 199 amino acids and mals. It is best known for its stimulation of lactation and its three disulfide-bonded loops and is the primary form in human regulatory roles in the growth and differentiation of the mam- circulation (Sinha, 1995). mary gland and in reproduction (Clarke and Bern, 1980; Sinha, Since abnormal levels of hPRL have been linked to a number 1995; Goffin et al., 2002, 2005). of disorders in the areas of pituitary function and reproduction While human PRL (hPRL) presents different isoforms as a (e.g., hyperprolactinemia, galactorrhea, infertility), it is one of result of pre- or post-translational modifications (e.g. glycosy- the hormones most frequently determined in routine clinical assays (Kirby et al., 1979; Morganti et al., 1996; Glezer et al., 2006). Consequently, there is a considerable need for highly Abbreviations: A600, units of absorbance at a wavelength of 600 nm; purified hPRL in the preparation of in vitro diagnostic reagents. CHO, Chinese hamster ovary; cIts, thermo-sensitive repressor of the PL pro- moter of bacteriophage ␭; CRS, Chemical Reference Standard; DsbA, a 21 kDa Although routine therapeutic applications of either hPRL or E. coli periplasmic protein; IPTG, isopropyl-beta-d-thiogalactopyranoside; its analogs/antagonists (Soares et al., 2006) have not yet been hGH, human growth hormone; hPRL, human prolactin; rhPRL, recombinant established, former studies indicate several possible applications ␭ ␭ human prolactin; PL, major leftward promoter of the bacteriophage ; LB, of this hormone (Soares et al., 2000), while more recent studies Luria–Bertani medium; Mr, relative molecular mass; PAGE, polyacrylamide-gel in rats have shown that PRL levels increase in response to stress, electrophoresis; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; ◦ helping to protect against acute stress-induced hypocalcemia tA, activation temperature (in C); tR, retention time ∗ Corresponding author. Tel.: +55 11 31339699; fax: +55 11 31339694. and gastric erosions (Fujikawa et al., 2004). Zhang et al. (2005), E-mail address: [email protected] (C.R.J. Soares). report that recombinant hPRL (rhPRL) promotes antitumor 0168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2007.08.038 28 C.R.J. Soares et al. / Journal of Biotechnology 133 (2008) 27–35 effects of human NK cells transferred to tumor-bearing SCID the vector containing the ␭PL promoter, but without the pres- mice. At present, different clinical trials on rhPRL applied to ence of the repressor protein, and under optimized temperature lactation induction are underway (www.clinicaltrials.gov). In conditions. view of the above, an increasing need for pure, biologically active, authentic hPRL can be anticipated. 2. Material and methods Several laboratories have reported expression of hPRL in dif- ferent prokaryotic or eukaryotic host cells and the strategies that 2.1. Plasmid constructions have been applied for the synthesis of this and other human pituitary hormones are described in a recent review (Ribela et The DNA sequence, including the restriction endonuclease al., 2003). An interesting strategy for obtaining authentic hPRL site Nde I, start codon, DsbA signal peptide, hPRL cDNA is bacterial periplasmic expression, a cheaper alternative com- coding for the native sequence of the hormone, stop codon pared to mammalian cells expression (Price et al., 1995) that, and Bam HI site, was obtained by polymerase chain reac- as far as we are aware, has only been described by our group tion (PCR) utilizing the plasmid p3SN8hPRL3 (Morganti (Morganti et al., 1998; Ueda et al., 2001; Soares et al., 2002). et al., 1998) as a template. The synthetic primers used for Periplasmic specific expression yields higher than 0.1 ␮gml−1 this PCR were: forward primer, including the Nde I site, −1 A600 were, however, never reported. Periplasmic secretion of DsbA signal peptide and 20 nt of the hPRL cDNA sequence recombinant proteins offers a multitude of advantages when (5-GAT-CGA-TCG-ATC-CAT-ATG-AAA-AAG-ATT-TGG- compared to other expression systems in Escherichia coli and in CTG-GCT-CTG-GCT-GGT-TTA-GTT-TTA-GCG-TTT-AGC- mammalian cells (e.g., CHO). Among these, we can mention: GCA-TCG-GCG-TTG-CCC-ATC-TGT-CCC-GGC-GG-3); low contaminants, low proteolytic activity and lower amounts reverse primer, including a C-terminal sequence of the hPRL or even absence of undesirable isoforms such as high molec- gene and the Bam HI site, downstream of the stop codon (5- ular weight, glycosylated, phosphorylated or cleaved forms. GAT-CGA-TCG-GAT-CCT-TAT-CAG-CAG-TTG-TTG-TTG- Comparing directly with cytoplasmic expression, the periplasm TTG-AT-3). After digestion of the DNA fragments obtained by contains only 4% of the total cell proteins, which makes purifi- PCR with the endonucleases Nde I and Bam HI, the fragments cation considerably less onerous. The cleavage in vivo of the were subcloned into the Nde I–Bam HI sites of a cassette signal peptide provides, moreover, the authentic N terminus of expression vector, the same utilized to obtain the hGH vector the target protein (i.e. without an extra methionine) while the (␭PL–DsbA–hGH) containing the ␭PL promoter and ampicillin oxidizing environment of the periplasm facilitates the proper resistance gene (Soares et al., 2003). The resulting hPRL vector folding of the protein (Makrides, 1996; Soares et al., 2003). was named ␭PL–DsbA–hPRL. Despite all the desirable features of this secretion system, some heterologous proteins fail to be successfully expressed in the 2.2. Bacterial strains and shake flask cultivation periplasm, limiting this technique to certain selected candidate products (Soares et al., 2006). E. coli RRI strain, carrying the low copy-number plas- In our laboratory, high periplasmic expression levels of mid pRK248cIts (Bernard and Helinski, 1979) encoding for human growth hormone (hGH) had been achieved by using a the thermo-sensitive transcription repressor (cIts) gene, was system based on the control of ␭PL promoter by the thermo- used for propagation and extraction of pRK248cIts. For hPRL sensitive cIts regulator (Soares et al., 2003). Indeed, this is one or hGH expression, the E. coli host strain W3110, obtained of the most widely used promoters for E. coli large-scale pro- from American Type Culture Collection (ATCC), was used. tein production. This promoter was chosen based on cost and Four transformed E. coli W3110 strains were used: two health/safety concerns since thermal induction is free of chemi- harboring only the expression vector (␭PL–DsbA–hPRL or cal inductors such as IPTG or nalidixic acid, which are expensive ␭PL–DsbA–hGH), selected on LB agar plates supplemented and potentially hazardous for personnel and the environment with 100 ␮g of amp ml−1, and two strains harboring the expres- and thus less attractive for large-scale production of human sion vector plus the repressor gene-bearing plasmid pRK248cIts, therapeutic proteins (Makrides, 1996; Soares et al., 2003). This selected on LB plates supplemented with 100 ␮g of amp and thermo-inducible promoter is activated at temperatures around 50 ␮g of tetracycline ml−1. Cultivation was carried out in 500 ml 42 ◦C and fully repressed at 30 ◦C. The major shortcomings of shake flasks containing 100 ml of Luria–Bertani (LB) medium. this temperature shift methodology are the problems commonly Each flask was inoculated with a single colony of E. coli cells associated with protein expression above 37 ◦C (e.g., proteoly- taken from LB agar plates and incubated on a rotary shaker at ◦ sis, degradation, aggregation) that can negatively influence the 30 C.
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