Production of human growth hormone by Lactococcus lactis

Journal of Bioscience and Bioengineering VOL. 109 No. 4, 322 – 324, 2010 www.elsevier.com/locate/jbiosc

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Production of human growth hormone by Lactococcus lactis Abelardo Margolles,1,⁎ José Antonio Moreno,1,† Lorena Ruiz,1 Belkis Marelli,2 Christian Magni,2 Clara G. de los Reyes-Gavilán,1 and Patricia Ruas-Madiedo1 Departamento de Microbiología y Bioquímica de Productos Lácteos, Instituto de Productos Lácteos de Asturias (CSIC), Carretera de Infiesto s/n 3300 Villaviciosa, Asturias, Spain 1 and Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET) and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531 (S2002LRK) Rosario, Argentina 2 Received 15 September 2009; accepted 6 October 2009 Available online 27 October 2009

A synthetic gene coding for human growth hormone was expressed in Lactococcus lactis. The presence of the recombinant protein was assayed and quantified using ELISA tests. Human growth hormone was detected at high concentrations and displayed a biological activity similar to the one shown by commercial human growth hormone. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Human growth hormone; Heterologous expression; Lactococcus lactis; Food-grade; Caco-2 proliferation]

Lactococcus lactis is a food-grade microorganism that has been extensively used in food fermentation for centuries, being consumed by humans mainly in dairy based products. Furthermore, several genomes of L. lactis have been sequenced (1, 2) and the genetics of different L. lactis strains are very well characterised nowadays (3). This has allowed the development of numerous expression systems that have been successfully used for heterologous protein expression in this host (4-6). Due to the harmlessness of this bacterium, L. lactis has been used for more than 10 years now as a delivery vehicle in mucosal vaccine research and other health-related applications (7), as well as a cell factory to produce heterologous proteins with important biotechnological uses (8, 9). Even human intervention studies have recently been carried out with a recombinant strain of L. lactis expressing human interleukin-10, demonstrating that mucosal delivery of therapeutic proteins by L. lactis is feasible in humans (10). In this context, the aim of this work was to evaluate the capacity of L. lactis to produce human growth hormone (hGH), as an attractive alternative to the classical expression systems using other recombinant microorganisms. Numerous studies have demonstrated the capacity of other bacterial hosts, such as Escherichia coli and Bacillus sp., to produce recombinant hGH (rhGH) in an active form, either using secretion systems able to generate milligrams of protein per litre of culture, or other strategies producing the recombinant protein intracellularly at very high levels, for instance as inclusion bodies (11-14), but to the

⁎ Corresponding author. Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Carretera de Infiesto s/n, PO 85, 3300 Villaviciosa, Asturias, Spain. Tel.: +34 985892131; fax: +34 985892233. E-mail address: [email protected] (A. Margolles). † Current address: Dep. Investigación Básica, Laboratorios Ordesa, Parc Científic de Barcelona, Edifici Hèlix, Carrer Baldiri Reixac 15-21, 08028 Barcelona, Spain.

best of our knowledge this has never been investigated before in a food-grade bacterium. A synthetic gene was synthesised taking into account the codon usage of the L. lactis strain IL1403 (1), yielding the mature form of hGH (GenBank accession no. 1224637). The gene was cloned into the expression vectors pNZ8048 (15) and pHPB22 (5), and the strain L. lactis NZ9000 was transformed following standard procedures. The only difference between the mature form of the human protein and the recombinant protein was that rhGH was synthesized with an Nterminal methionine, coded in the forward primer, thus yielding a 192 amino acid polypeptide chain. Strains, vectors and primers used in this work are shown in Table 1. L. lactis NZ9000 cell-free extracts from cells containing pNZ vectors were obtained basically as described by Margolles and de los Reyes-Gavilán (8) with minor modifications. L. lactis NZ9000 cells were grown in 500 ml of M17 medium (Oxoid LTD, Basingstoke, Hampshire, UK) supplemented with 0.5% glucose (GM17) and 5 μg ml−1 chloramphenicol. Incubations were carried out at 30 °C to an OD600 of 0.5 and, at this point, transcription was triggered by adding 0.1% (v/v) supernatant from a culture of the nisin-producing strain L. lactis NZ9700. Then the cells were incubated for 75 min at 30 °C (which resulted in an optical density of about 1.1), harvested by centrifugation, washed twice with phosphate-buffered saline (Dulbecco's PBS, Sigma Chemical Co., St. Louis, MO), and resuspended in 10 ml of the same buffer. The protease inhibitor Pefabloc (0.4 mM; Roche Applied Sciences, Mannheim, Germany) was included in the solution and the cells were disrupted by passage twice through a Cell Disrupter (Constant Systems LTD, Daventry, UK). Unbroken cells and cell debris were removed from the solution by ultracentrifugation at 125,000 g for 20 min, glycerol was added to the supernatant to reach a concentration of 2.5% (v/v) and the protein solution was frozen in liquid nitrogen until use. The same procedure was used to obtain L.

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TABLE 1. Strains, vectors and primers used in this study. Strains L. lactis NZ9000 L. lactis NZ9700 Vectors pNZ8048 pHPB22 pNZ-hGH pCit-hGH Cloning primers fn-hGH rn-hGH

fc-hGH rc-hGH

Characteristics

Reference

L. lactis MG1363 pepN::nisRK Nisin producer

17 17

Gene expression vector with nisin inducible PnisA promoter, Cmr pAK80-derived plasmid containing the 0.4-kb cit promoter region from L. lactis CRL264 pNZ8048 derivative containing the rhGH gene pHPB22-derivative containing the rhGH gene under control of the Pcit promoter region

15

CTC TAA CAT GTT TCC AAC AAT TCC TTT AT Forward cloning primer for pNZ8048, BspLU11I site TGC CGA CTC TAG AGG ATC CTT ATT A Reverse cloning primer for pNZ8048, BamHI and BglII sites CAC TCT CCA TGG TTC CAA CAA TTC C Forward cloning primer for pHPB22 vector TGC TTT GTC GAC TGC TGC TTT TTG Reverse cloning primer for pHPB22 vector

5 This work This work

This work This work

This work This work

lactis NZ9000 cell-free extracts from cells containing the vector pCit-hGH grown for 12 h in GM17 with 5 μg ml−1 erythromycin (OD600 about 1.5), in which the induction of the gene from the promoter Pcit was triggered by the lactic acid produced in the batch culture during fermentation (5). Total protein quantification was carried out with the BCA method (Pierce, Rockford, IL). The amount of rhGH present in the lactococcal extracts was determined using the Human GH ELISA BioSource kit (BioSource Europe, S.A., Nivelles, Belgium) following the manufacturer's instructions. The correlation coefficient (R2 ) of the corresponding standard regression equation was 0.95. Serial dilutions of the extracts, each analysed in duplicate, were measured. The control extract, from L. lactis NZ9000 cells containing an empty vector, did not contain detectable amounts of hGH. Remarkably, the amount of rhGH was very different depending on the expression system used, being close to 1% for protein extracts coming from cells with the pNZhGH vector (9.4 ± 1.7 μg mg−1 total protein) and about 30 times less from cells containing the pCit-hGH vector (0.31 ± 0.06 μg mg−1 total protein). Previous studies have demonstrated that hGH stimulates proliferation of human intestinal cell lines (16). To test if this biological activity was retained by the recombinant protein produced in L. lactis, a mitogenic assay using the epithelial intestinal Caco-2 cells (ECACC 86010202, purchased by the European Collection of Cell Cultures, Salisbury, UK) was carried out. Caco-2 cells were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with non-essential amino acid solution, 20% heatinactivated fetal bovine serum, and 50 μg ml−1 gentamicin, 25 μg ml−1 streptomycin–penicillin, and 1.25 μg ml−1 amphotericin B. All reagents were purchased from Sigma and incubations took place at 37 °C in 5% CO2 in a SL Waterjacked CO2 Incubator (Sheldon Mfg., Inc., Cornelius, OR). Culture media were changed every 2 days and the cell lines were trypsinized weekly following standard procedures. For experiments, Caco-2 cells were used at pass 50. For cell proliferation assays, the cell lines were seeded in 96-well fluorometry validated plates at a concentration of 7 × 104 cells ml−1, and incubated for 1 day. Afterwards, 10 μl of cell-free lactococcal extracts diluted in supplemented DMEM were added daily for 3 consecutive days, the final one added after changing the culture media. The total protein concentration of the lactococcal extracts ranged from 1 to 100 μg ml−1 total protein content, since higher amounts of extract than those indicated above were toxic for the Caco-2 cells and inhibited their proliferation (data not shown). These total protein concentrations correspond to approximately 10–

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1000 ng ml−1 of rhGH in the extracts coming from cells containing the vector pNZ-hGH, and to 0.3–30 ng ml−1 of rhGH in the extracts coming from cells containing the vector pCit-hGH. Therefore, from 0.1 to 10 ng, or from 0.003 to 0.3 ng of rhGH, respectively, were added daily to the wells. As a positive control, several amounts of hGH standard (Biosource) (from 0.003 to 10 ng, added daily diluted in supplemented DMEM for 3 consecutive days, the final one added after changing the culture media), were employed. Plates with lactococcal extracts and standards were incubated in a Heracell® 240 incubator (Thermo Electron LDD GmbH, Langenselbold, Germany) at 37 °C in 5% CO2, and cell proliferation was determined by using the BrdU-HTS Cell Proliferation Assay (Calbiochem, Merck, Darmstadt, Germany) following the manufacturer's instructions. This method measures the DNA of new synthesis in cells under active proliferation using fluorescence probes. Fluorescence emission was measured in a Cary Eclipse Fluorescence Spectrophotometer (Varian Ibérica S.A., Madrid, Spain). Measurements were carried out in duplicate for each concentration/lactococcal extract type and the relative fluorescence was calculated with respect to that of the negative control (without lactococcal extract added). One-way ANOVA and the LSD mean comparison test were performed in order to establish significant differences in the ability of the cell-free lactococcal extracts to modify the proliferation of the cell lines. Results of the mitogenic analysis are shown in Fig. 1. The samples containing rhGH were able to promote a higher proliferation of Caco-2 cells than the samples not containing it, although significant differences, comparable to the ones produced by commercial hGH, were only observed for the samples in which the induction of the gene was triggered by nisin. This is probably due to the higher concentration of the recombinant hormone present in the cell-free extract coming from cells containing the vector pNZ-hGH. In relation to this, induction times shorter than 75 min yielded extracts with no significant mitogenic activity (data not shown). In summary, in this work we have been able to produce rhGH in L. lactis. rhGH was produced in this host at high levels and was able to retain biological activity by inducing the proliferation of intestinal cell lines in vitro. This is the first work reporting the production of this human protein in lactic acid bacteria.

FIG. 1. Proliferation of Caco-2 cells in the presence of lactococcal extracts containing rhGH or human GH standard. The relative fluorescence emitted was calculated with respect to that obtained in the Caco-2 cells cultured in media without lactococcal extract (negative control), arbitrarily set to 100. The proliferation results correspond to the addition of lactococcal extracts containing approximately 0.3 ng of rhGH (pNZhGH) or about 0.009 ng of rhGH (pCit-hGH). The positive control (GH standard) corresponds to the addition of 0.3 ng of hGH standard, since results of the addition of 0.009 ng of hGH standard did not significantly differ from those of the lactococcal extract in which the rhGH was absent (pNZ8048, strain containing the empty plasmid, dotted line). Bars that do not share the same letter are significantly different (p b 0.05).

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MARGOLLES ET AL. ACKNOWLEDGMENTS

This work was financially supported by the Plan Regional de I + D from Asturias (project PB02-023), by FEDER funds and by the Spanish Plan Nacional de I + D + i (project AGL2006-03336). We gratefully thank Prof. Oscar Kuipers and NIZO Food Research for providing L. lactis strains NZ9000 and NZ9700 and plasmid pNZ8048. References 1. Bolotin, A., Wincker, P., Mauger, S., Jaillon, O., Malarme, K., Weissenbach, J., Ehrlich, S. D., and Sorokin, A.: The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403, Genome Res., 11, 731–753 (2001). 2. Wegmann, U., O'Connell-Motherway, M., Zomer, A., Buist, G., Shearman, C., Canchaya, C., Ventura, M., Goesmann, A., Gasson, M. J., Kuipers, O. P., van Sinderen, D., and Kok, J.: Complete genome sequence of the prototype lactic acid bacterium Lactococcus lactis subsp. cremoris MG1363, J. Bacteriol., 189, 3256–3270 (2007). 3. Kok, J., Buist, G., Zomer, A. L., van Hijum, S. A., and Kuipers, O. P.: Comparative and functional genomics of lactococci, FEMS Microbiol. Rev., 29, 411–433 (2005). 4. Llull, D. and Poquet, I.: New expression system tightly controlled by zinc availability in Lactococcus lactis, Appl. Environ. Microbiol., 70, 5398–5406 (2004). 5. Martín, M. G., Sender, P. D., Peirú, S., de Mendoza, D., and Magni, C.: Acidinducible transcription of the operon encoding the citrate lyase complex of Lactococcus lactis Biovar diacetylactis CRL264, J. Bacteriol., 186, 5649–5660 (2004). 6. Mierau, I. and Kleerebezem, M.: 10 Years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis, Appl. Microbiol. Biotechnol., 68, 705–717 (2005).

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