Curr Microbiol DOI 10.1007/s00284-013-0500-9
Surface Display of Human Growth Hormone on Bacillus subtilis Spores for Oral Administration Chaoqun Lian • Yang Zhou • Fan Feng • Liang Chen • Qi Tang • Qin Yao • Keping Chen
Received: 24 September 2013 / Accepted: 15 October 2013 Ó Springer Science+Business Media New York 2013
Abstract Human growth hormone (hGH) is the major and important hormone component of human being. At present, hGH for clinical uses is mostly produced in Escherichia coli, which requires costly denaturation and refolding to recover functionality. To obtain long-term bioactive hormone, we used hGH as a foreign gene and constructed a recombinant plasmid pJS700-hGH which carries a recombinant gene cotC-hgh with an enterokinase site under the control of cotC promoter. Plasmid pJS700hGH was transformed into Bacillus subtilis by double crossover and an amylase-inactivated mutant was produced. After spore formation, Western blot and fluorescence immunoassay were used to monitor hGH surface expression on spores. Oral administration to silkworm with spores displaying hGH further showed that the recombinant spores may have potential ability to be digested and absorbed into the silkworm’s hemolymph due to both the resistant characters of spores and the addition of enterokinase site.
C. Lian (&) F. Feng K. Chen School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, Jiangsu, People’s Republic of China e-mail: [email protected] K. Chen e-mail: [email protected] C. Lian Department of Clinical Laboratory, Bengbu Medical College, 2600 Donghai Road, Bengbu 233030, Anhui, People’s Republic of China Y. Zhou L. Chen Q. Tang Q. Yao Institute of Life Sciences, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, Jiangsu, People’s Republic of China
Introduction The Human growth hormone (hGH) is a 22-kDa singlechain polypeptide of 191 amino acids [14]. It is produced by the pituitary gland and is required for normal growth of body and bones. Besides its role in growth and development, it can help improve overall health, energy levels, muscle mass and metabolism, and its absence or low production in children and teenagers (mainly caused by genetic mutations) is partly to be blamed for causing hypopituitary pediatric dwarfism [4]. A common clinical treatment for this disorders is hGH injections. However, it is very expensive [30], representing a huge barrier to patients. Endogenous hGH is a non-glycosylated protein; moreover, recombinant forms of hGH have been extensively produced in prokaryotic expression systems [1]. The production of recombinant hGH in transgenic bacterial systems has considerably reduced the overall treatment costs of dwarfism and other related growth deficiencies. Although currently commercially available hGH is mainly produced by prokaryotic expression systems, this system still has some drawbacks. First, the recombinant hGH is overexpressed in aggregated, insoluble inclusion bodies, which later requires costly denaturation and refolding to recover functionality. Moreover, the outer cell membrane of most Gram-negative bacteria, e.g., Escherichia coli, contains the highly immunogenic lipopolysaccharides (LPS), generally referred to as endotoxins, traces of which have to be removed from proteins that need to be injected into humans and animals [7]. Therefore, the development of an alternative method of producing low-cost hGH in large quantity is desired. The Gram-positive bacterium Bacillus subtilis (B. subtilis) is considered as a GRAS organism (generally recognized as safe). In contrast to E. coli, the use of B. subtilis
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for the production of food products is highly favored over the use of E. coli. Furthermore B. subtilis is a rod-shaped, aerobic bacterium, and it can form spores, a metabolically quiescent and extremely resistant cell type, as a response to harsh environmental conditions [9, 12, 28, 29]. The peculiar structures, including a proteinaceous bilayer coat and the low content of water, offer unique resistance properties to extremes of temperature, dessication, and exposure to solvents and other noxious chemicals. Bacillus subtilis spore is encased by a multilayered coat protein structure, which is critical for resistance properties as well as germination [12]. They provide spore mechanical integrity and exclude large toxic molecules and chemicals from invading the spore core [9, 12, 19]. Detailed genetic and morphological studies have shown that the proteinaceous nature of the coat suggests the potential possibility of using its structure components as fusion partners for the expression of heterologous proteins on the spore surface. Consequently, previous researches indicated that B. subtilis spore was a powerful vehicle for delivery of heterologous antigens or some bioactive molecules [6, 20, 26]. Preclinical tests in animal models are essential for evaluating the therapeutic effects of drug candidates for further development. Mammals, such as mouse, rabbit, and so on, are commonly used as drug-screening models to examine the pharmacodynamics of chemicals. While the use of mammals for drug development often cause some problems with regard to ethical issues [2]; the development of invertebrate animals as drug-screening models will overcome these problems. Invertebrate animals such as Romalea microptera [16], Caenorhabditis elegans [24], and Drosophila melanogaster [3] have been generally used in bacterial infection models. Among those modes, silkworms got a huge attention because those exclusive properties, including low cost, convenient feeding, effectiveness of the drug-screening, and involved in the use of mammals. Therefore, we used the silkworm for evaluating the effects of compounds. In this study, we used outer coat component cotC as a fusion partner for surface display of hGH on B. subtilis spores. A recombinant B. subtilis strain, which displayed hGH on the spore surface, has been successfully constructed and identified with hGH-specific antibody; in addition, the bioactive effect was examined by oral administration of silkworm with spores displaying hGH.
Materials and Methods Materials Bacillus subtilis 168 (trp-) was obtained from Bacillus Genetic Stock Center, Department of Biochemistry, The
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Ohio State University. The expression vector pET-30a(?) and E. coli strains JM109 and BL21(DE3) were obtained from Novagen (CA, USA). hGH rabbit polyclonal antibody and the vector pCR4-TOPO containing the hgh gene were purchased from Proteintech Group (Shanghai, China). High-fidelity LA Taq polymerase, restriction enzymes, T4 DNA ligase, and the subcloning vector pMD18-T were purchased from TaKaRa Bio Inc (Dalian, China). Chemicals are all from Sigma (MO, USA) or a domestic provider in China if not stated otherwise. Preparation and transformation of B. subtilis strain 168 (trp-)-competent cells were performed as previously described [33]. QINGSONG was used for the oral administration experiment as a silkworm strain reared and kept in our laboratory. Cloning of Human Growth Hormone Gene The hgh-specific primers, hgh-F: 50 -GGCCATGGTTCCCA ACCATTCCCTTATC-30 (NcoI site is underlined) and hgh-R: 50 -CGAGCTCTTAGAAGCCACAGCTGCCCTC-30 (SacI site is underlined), were designed to amplify an 573-bp hgh gene (GenBank accession no. BC 075012) from the vector pCR4-TOPO. The PCR product was ligated into the pMD18-T vector to prepare plasmid pMD18-hGH. Sequencing of the cloned PCR product was carried out by Sangon Biotech (Shanghai) Co., Ltd., China. Construction of Plasmid pJS700-hGH and Recombinant B. subtilis To obtain an integration of the cotC-hgh fusion gene with an enterokinase site (Asp-Asp-Asp-Asp-Lys) at the B. subtilis amyE, a recombinant plasmid for double crossover with B. subtilis chromosome was constructed. First, plasmid pMD18-hGH and pET30a(?) were digested, respectively, with NcoI and SacI, and purified with Omega BioTek Gel Extraction Kit. Next, the purified hgh gene was inserted into prepared pET30a(?) to construct recombinant plasmid pET30a-hGH, in which between the upstream of the hgh gene and the downstream of Restriction Enzyme cutting site KpnI included an enterokinase site. Finally, we used the restriction endonuclease KpnI and SacI to digest the plasmid pET30a-hGH and pJS700, and inserted the hgh gene into pJS700, resulting in recombinant plasmid pJS700-hGH; in which the upstream and downstream of the region erythromycin (Em)-cotC-hGH was homologous to B. subtilis amyE, as was verified by sequencing [23]. Plasmid pJS700-hGH was digested with BglII, and the resulted linear fragment containing Em-cotC-hGH gene with an enterokinase site (Asp-Asp-Asp-Asp-Lys) was purified with the Gel Extraction Kit and then transformed into the B. subtilis strain 168 (trp-)-competent cells. The
C. Lian et al.: Human Growth Hormone on Bacillus subtilis Spores
transformed cells were incubated in 2 ml SOB medium and cultured at 37 °C overnight with gentle shaking (*80 rpm) for 40 min, followed by spraying onto LB plate containing 0.4 lg/ml erythromycin. Plates were incubated at 37 °C overnight, Emr colonies were selected, and a colony with cotC-hgh integrated at the B. subtilis amyE locus was identified by analysis of amylase activity and then confirmed by PCR. Screening of Mutants with CotC-hGH Integrated at amyE Locus The integration of cotC-hgh at amyE locus will disrupt amyE gene (GenBank accession no. NP_388186.2) causing amylase-negative phenotype on LB plates containing 1 % starch [33]. After incubation at 37 °C overnight, the plates were stained by iodine to examine the amylase activity. A blue color was produced by starch–iodine reaction in the presence of cotC-hgh fusion in the B. subtilis chromosome. Otherwise, no blue color was observed at the B. subtilis 168 (trp-) control when iodine was added, since the expression of amyE resulted in hydrolysis of the starch in the plate. Chromosomal DNA was extracted from both the B. subtilis 168 (trp-) control and cotC-hgh transformants for site-directed PCR. Primers amyE-F: 50 -ATTGCTCGGGCT GTATGACTGG-30 and amyE-R: 50 -GTTACACCATCAC TGTTCGTTCCTT-30 will amplify about 1-kbp wild-type fragment and a 4.3-kb integrated fragment. Primer pair hgh-F and hgh-R was used to detect the correct insertion of the hgh gene. Expression of CotC-hGH on B. subtilis Spore Surface Sporulation of wild-type B. subtilis (trp-) and recombinant strain cotC-hGH was induced in Difco sporulation medium (DSM) by the exhaustion method as previously described [31]. Cultures were harvested 48 h after the initiation of sporulation. Spores were collected, washed several times, and performed by lysosome treatment as described by Nicholson and Setlow to break any residual sporulating cells [31]. Phenylmethylsulfonylfluoride (PMSF, 0.05 M) was included to inhibit proteolysis. Spore coat proteins in suspensions at high density ([1 9 1010 spores/ml) were extracted from B. subtilis (trp-) spores and the cotC-hGH spores using an SDS–DTT extraction buffer as previously described [33]. Western Blot Analysis and Fluorescence Immunoassay Extracted coat proteins were mixed with SDS-PAGE loading buffer and separated on a 12 % polyacrylamide gel at room temperature. Electrophoresis was at 80 V for
30 min and then at 120 V for 150 min. The gel was stained with 0.25 % Coomassie blue R-250 for 1 h and then destained for 12 h using a mixture of 10 % acetic acid and 5 % ethanol. To confirm that cotC-hGH was expressed on the B. subtilis spore surface, the proteins in the gel were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore) for Western blotting using hGH-specific antibody at a dilution of 1:1,000. Immunoreactive proteins were visualized using goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (HRP) as described [13]. In addition, B. subtilis spores cultured in DSM were collected and used for fluorescence immunoassay using hGH-specific antibody and FITC-conjugated goat antirabbit IgG. Fluorescence was examined by excitation at 488 nm with Confocal laser scanning microscope (CLSM, Leica, Germany). Oral Administration of Silkworm with Spores and Hemolymph Protein Extraction for Western Blot Analysis In order to verify the hGH protein that displayed on the B. subtilis spore surface and whether it could be digested and absorbed, the test that the oral administration of silkworm with B. subtilis spores was performed in vivo. Newly molted fifth instar healthy larvae was starved for 6 h, and then inoculated individually through oral administration with 10 ll suspension spores of concentration of 2 9 1010 spores/ml once day [25]. After 5 days normal rearing, the hemolymph in silkworm was collected for protein concentration measurement, which was determined spectrophotometrically at 595 nm using bovine serum albumin for the standard curve determinations [5]. Hemolymph samples (5 larvae, 200 ll hemolymph) were collected from severed third prolegs of fifth instar larvae. The larvae were reared on mulberry leaves in laboratory conditions (25 ± 1 °C, 65 % room humidity and 14-h light:10-h dark). There were 90 larvae averagely split into 3 groups: one control group (C), another wild-type spore treatment group (WT), and the final transgenic spore treatment group (TG). Proteins extracted from silkworms’ hemolymph in each group were fractionated on 12 % denaturing polyacrylamide gels, and then the Western blot method was performed as the same above.
Results Construction of Integrative Vector Containing cotChgh Gene with an Enterokinase Site The strategy to obtain recombinant B. subtilis spores expressing hGH with an enterokinase site on their surface
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was based on the use of the cotC gene and its promoter for the construction of translational fusions and on chromosomal integration of the cotC-hgh gene with an enterokinase site inserting into the coding sequence of the amyE gene. Then the hgh gene with a enterokinase site was obtained from the digestion of plasmid pET-30a-hGH with the restriction endonuclease KpnI and SacI, and cloned at the 30 -end of cotC open reading frame in plasmid pJS700 to generate the recombinant plasmid pJS700-hGH, in which hGH and cotC were co-expressed under the control of cotC promoter (Fig. 1a). The obtained fusion gene was integrated into the B. subtilis 168 chromosome at the amyE locus by a double crossover event (Fig. 1b). Identification of cotC-hpi Integrated in B. subtilis Chromosome The recombinant B. subtilis having integrated cotC-hgh into chromosome at the amyE locus by double crossover was first analyzed by amylase activity. The integration of cotC-hgh at amyE locus interrupted the expression of amylase. As a result, no transparent halo was observed around the recombinant clones on a starch-containing plate stained by iodine potassium iodide (Fig. 2a). It was indicated that the recombinant gene cotC-hgh was successfully inserted into B. subtilis chromosome. But the wild-type B. subtilis produced a transparent halo around the clones due to the expression of amylase (Fig. 2a). Disruption of amyE in B. subtilis chromosome and correct insertion of cotChgh at the amyE locus were further confirmed by PCR with four primer pairs. Primer pair amyE-F/amyE-R amplified a 1,098-bp (amylase gene) and 3,800-bp (fusion gene) fragment from wild-type B. subtilis (WT) and transgenic B. subtilis (TG) chromosome, respectively. Moreover, primer pairs hgh-F/hgh-R, amyE-F/hgh-R, and hgh-F/amyE-R produced fragments around 1.8 kb, 4.3 kb, and 2.3 kb on the transgenic B. subtilis chromosome, respectively. However, no PCR product was observed in wild-type B. subtilis chromosome (Fig. 2b). Display of hGH on Recombinant Spores To confirm that hGH was successfully expressed on recombinant B. subtilis spores, hGH-specific antibody was used to perform Western blot analysis. The result showed that a 30.8 kDa clear band was detected in the extraction from recombinant spores (Fig. 3, Lane 2). Since the part of cotC gene of B. subtilis encodes a 66 amino acid protein with a predicted molecular mass of 8.8 kDa [8], and the ORF of hgh encodes a 191 amino acid protein with a molecular mass of 22 kDa, the Western blot analysis suggested that the recombinant fusion protein was expressed with expected molecular mass. However, no similar band
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Fig. 1 Cloning strategy. a The flow chart of plasmid pJS700-hGH c construction. amyE 50 -end and amyE 30 -end are integrative fragments from amylase gene of B. subtilis 168 (trp-); Emr, erythromycinresistant site; cotC, a B. subtilis spore coat protein-encoding gene. b The strategy for the chromosomal integration of the cotC-hGH gene fusions. Arrows indicate the positions of primer pairs used in the confirmation of the hgh gene insertion in the mutant strain and wild type
in the extraction proteins from wild-type B. subtilis spores was detected (Fig. 3, Lane 1). Therefore, the result indicated that fusion protein cotC-hGH was expressed in the spore correctly. In order to verify that hGH was displayed on the spore surface, fluorescence immunoassay was performed with rabbit anti-hGH primary antibodies and FITC-conjugated goat anti-rabbit secondary antibodies. Bright green fluorescence could be observed on the surface of recombinant spores (Fig. 4) with CLSM, but no fluorescence signal was observed on the surface of wild-type B. subtilis spores (Control). This demonstrated that hGH was expressed on the surface of recombinant spores. Oral Administration of Silkworm with Spores Groups of silkworm were inoculated by the oral administration with recombinant spores of hGH-expressing strain, isogenic control strain and double-distilled water. In order to confirm that the hGH protein displayed on the recombinant spores could be digested and absorbed into the hemolymph of silkworm for the addition of enterokinase site (Asp-Asp-Asp-Asp-Lys), proteins extracted from silkworms’ hemolymph in each group were fractionated on 12 % denaturing polyacrylamide gels, and then the Western blot method was performed using previously prepared hGH-specific antibody. As shown in Fig. 5, a 22-kDa band was detected in the extracts from the hemolymph of TGtreated silkworm, while no similar band in the WT-treated group was detected, indicating that the hGH protein displayed on the recombinant spores could be digested and absorbed into the hemolymph of silkworm. In addition, this also demonstrated that the hgh gene was successfully displayed on the surface of B. subtilis spores.
Discussion The bacterial surface display technology has been attracting a great deal of attention due to their applications in the fields of vaccine, drug delivery vehicles, whole-cell biocatalysts, biosensors, environmental remediation, and biomolecule screening platforms [22]. Among various microbial cell surface display systems presently available,
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the newly developed spore surface display offers additional advantages such as higher stability and safety. Compared to other Bacillus species such as Bacillus anthracis [27] and Bacillus thuringiensis [32], B. subtilis has more advantages in developing spore surface display system due to the detailed knowledge of its spore structure [12]. B. subtilis spore display system is based on the construction of gene fusion between heterologous DNA and a B. subtilis gene coding for a component of the spore coat, where the spore coat protein has been shown to be located on its outside surface [12, 15]. So far three different spore coat proteins namely cotB, cotC, and cotG have been used [15, 34]. Among the three proteins, the content of cotC is up to 50 % [21], which means it should be a ideal fusion partner for expressing heterologous proteins. In this paper, we displayed hGH on the B. subtilis spore surface by fusion hgh to the C-terminal of cotC. This improvement may be contributed by the resistant characters of B. subtilis spores. It was reported that B. subtilis spores could survive in Fig. 2 Identification of the transgenic B. subtilis. a Identification of transgenic B. subtilis by amylase activity analysis. 1 Transgenic strains and wild-type B. subtilis growing on LB plate containing 1 % starch. 2 The plate stained with iodine. The insertion of cotC-hgh destroyed amyE and made the strain deficient in amylase, while the wild-type strain showed a big white halo around clones due to the secretion of amylase. b PCR analysis with different primer pairs. M, 250-bp DNA ladder marker; WT wild-type B. subtilis; TG transgenic B. subtilis with cotC-hgh insertion; primer pairs used in PCR are labeled above
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gastric environment, reach the intestinal tract, and germinate to vegetative cells in vivo. Results obtained from PCR, Western blot, amylase activity analysis, and immunofluorescence microscopy demonstrated that the cotC-hGH fusion protein was correctly exposed on the spore surface. Noteworthily, we found that the protein hGH we got was solube; therefore, the product should have a competitive edge on the future market. The study reported here is the first to describe hGH displayed on the surface of spores so far. To evaluate the effect of an enterokinase site in the fusion protein cotC-hGH, we used silkworms as a model. Immunoblot analysis of hemolymph in silkworm by oral administration with B. subtilis spores implied that the hGH protein displayed on the spores surface may be digested and absorbed into the silkworm hemolymph. This change may be contributed by the addition of enterokinase site, the resistant properties of B. subtilis spore, and the simple intestinal structure of silkworm.
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Generally, the use of mammals for drug development is expensive and highly problematic with regard to ethical issues. Currently, the similarities in the pharmacokinetics of drug between silkworms and mammals, including the mechanisms of chemical absorption, distribution, metabolism, and excretion have been widely reported [10, 11, 17, 18]. Moreover, the silkworm body size is large enough for hemolymph preparations and organ isolation, which are essential for studying the pharmacodynamics of drugs in animal bodies. In addition, it is far less costly to rear silkworms than mammals, and a large number of larvae can
Fig. 3 Western blot analysis of proteins extracted from B. subtilis spores. Fusion protein cotC-hGH was detected by hGH-specific antibody. Lane 1 wild-type B. subtilis spore proteins. Lane 2 recombinant B. subtilis spore proteins
be maintained in a small space. Screening of therapeutic agents can be easily performed with a large number of individual silkworms without the same ethical concerns involved in the use of mammals. Thus, we will further choose the use of silkworms as model animals for evaluating the therapeutic effects of cotC-hGH.
Fig. 5 Western blot analysis of proteins extracted from hemolymph in silkworm after oral administration with B. subtilis spores. Lane 1 hemolymph proteins in silkworm oral administrated with recombinant B. subtilis spores; lane 2 hemolymph proteins in silkworm oral administrated with wild-type spores; and lane 3 hemolymph proteins in silkworm oral administrated with ddH2O
Fig. 4 Immunofluorescent detection of recombinant spores. Bright-field images and immunofluorescent images of wild-type (control) and recombinant B. subtilis spores (10 9 100 oil immersion lens)
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Taken together, this study has displayed hGH on B. subtilis spores, and oral administration in silkworm suggested that the hGH protein displayed on the recombinant spores may be absorbed into its hemolymph, which laid a perfect foundation for evaluating the effect of cotC-hGH in the near future. Acknowledgments We honestly thank Professor Degang Ning for kindly gifting plasmid pJS700, and Professor Hengchuan Xia for language revision of the text. This study was supported by the National Basic Research Program of China ‘‘973’’ under Grant No. 2012CB114604, the Natural Science Research General Project of Education Office of Anhui Province (No. KJ2012B108), and Postgraduate Research and Innovation Project of Jiangsu Province (Nos. CXZZ12_0703 and CXZZ13_0699). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Surface Display of Human Growth Hormone on Bacillus subtilis Spores for Oral Administration Chaoqun Lian • Yang Zhou • Fan Feng • Liang Chen • Qi Tang • Qin Yao • Keping Chen
Received: 24 September 2013 / Accepted: 15 October 2013 Ó Springer Science+Business Media New York 2013
Abstract Human growth hormone (hGH) is the major and important hormone component of human being. At present, hGH for clinical uses is mostly produced in Escherichia coli, which requires costly denaturation and refolding to recover functionality. To obtain long-term bioactive hormone, we used hGH as a foreign gene and constructed a recombinant plasmid pJS700-hGH which carries a recombinant gene cotC-hgh with an enterokinase site under the control of cotC promoter. Plasmid pJS700hGH was transformed into Bacillus subtilis by double crossover and an amylase-inactivated mutant was produced. After spore formation, Western blot and fluorescence immunoassay were used to monitor hGH surface expression on spores. Oral administration to silkworm with spores displaying hGH further showed that the recombinant spores may have potential ability to be digested and absorbed into the silkworm’s hemolymph due to both the resistant characters of spores and the addition of enterokinase site.
C. Lian (&) F. Feng K. Chen School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, Jiangsu, People’s Republic of China e-mail: [email protected] K. Chen e-mail: [email protected] C. Lian Department of Clinical Laboratory, Bengbu Medical College, 2600 Donghai Road, Bengbu 233030, Anhui, People’s Republic of China Y. Zhou L. Chen Q. Tang Q. Yao Institute of Life Sciences, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, Jiangsu, People’s Republic of China
Introduction The Human growth hormone (hGH) is a 22-kDa singlechain polypeptide of 191 amino acids [14]. It is produced by the pituitary gland and is required for normal growth of body and bones. Besides its role in growth and development, it can help improve overall health, energy levels, muscle mass and metabolism, and its absence or low production in children and teenagers (mainly caused by genetic mutations) is partly to be blamed for causing hypopituitary pediatric dwarfism [4]. A common clinical treatment for this disorders is hGH injections. However, it is very expensive [30], representing a huge barrier to patients. Endogenous hGH is a non-glycosylated protein; moreover, recombinant forms of hGH have been extensively produced in prokaryotic expression systems [1]. The production of recombinant hGH in transgenic bacterial systems has considerably reduced the overall treatment costs of dwarfism and other related growth deficiencies. Although currently commercially available hGH is mainly produced by prokaryotic expression systems, this system still has some drawbacks. First, the recombinant hGH is overexpressed in aggregated, insoluble inclusion bodies, which later requires costly denaturation and refolding to recover functionality. Moreover, the outer cell membrane of most Gram-negative bacteria, e.g., Escherichia coli, contains the highly immunogenic lipopolysaccharides (LPS), generally referred to as endotoxins, traces of which have to be removed from proteins that need to be injected into humans and animals [7]. Therefore, the development of an alternative method of producing low-cost hGH in large quantity is desired. The Gram-positive bacterium Bacillus subtilis (B. subtilis) is considered as a GRAS organism (generally recognized as safe). In contrast to E. coli, the use of B. subtilis
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for the production of food products is highly favored over the use of E. coli. Furthermore B. subtilis is a rod-shaped, aerobic bacterium, and it can form spores, a metabolically quiescent and extremely resistant cell type, as a response to harsh environmental conditions [9, 12, 28, 29]. The peculiar structures, including a proteinaceous bilayer coat and the low content of water, offer unique resistance properties to extremes of temperature, dessication, and exposure to solvents and other noxious chemicals. Bacillus subtilis spore is encased by a multilayered coat protein structure, which is critical for resistance properties as well as germination [12]. They provide spore mechanical integrity and exclude large toxic molecules and chemicals from invading the spore core [9, 12, 19]. Detailed genetic and morphological studies have shown that the proteinaceous nature of the coat suggests the potential possibility of using its structure components as fusion partners for the expression of heterologous proteins on the spore surface. Consequently, previous researches indicated that B. subtilis spore was a powerful vehicle for delivery of heterologous antigens or some bioactive molecules [6, 20, 26]. Preclinical tests in animal models are essential for evaluating the therapeutic effects of drug candidates for further development. Mammals, such as mouse, rabbit, and so on, are commonly used as drug-screening models to examine the pharmacodynamics of chemicals. While the use of mammals for drug development often cause some problems with regard to ethical issues [2]; the development of invertebrate animals as drug-screening models will overcome these problems. Invertebrate animals such as Romalea microptera [16], Caenorhabditis elegans [24], and Drosophila melanogaster [3] have been generally used in bacterial infection models. Among those modes, silkworms got a huge attention because those exclusive properties, including low cost, convenient feeding, effectiveness of the drug-screening, and involved in the use of mammals. Therefore, we used the silkworm for evaluating the effects of compounds. In this study, we used outer coat component cotC as a fusion partner for surface display of hGH on B. subtilis spores. A recombinant B. subtilis strain, which displayed hGH on the spore surface, has been successfully constructed and identified with hGH-specific antibody; in addition, the bioactive effect was examined by oral administration of silkworm with spores displaying hGH.
Materials and Methods Materials Bacillus subtilis 168 (trp-) was obtained from Bacillus Genetic Stock Center, Department of Biochemistry, The
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Ohio State University. The expression vector pET-30a(?) and E. coli strains JM109 and BL21(DE3) were obtained from Novagen (CA, USA). hGH rabbit polyclonal antibody and the vector pCR4-TOPO containing the hgh gene were purchased from Proteintech Group (Shanghai, China). High-fidelity LA Taq polymerase, restriction enzymes, T4 DNA ligase, and the subcloning vector pMD18-T were purchased from TaKaRa Bio Inc (Dalian, China). Chemicals are all from Sigma (MO, USA) or a domestic provider in China if not stated otherwise. Preparation and transformation of B. subtilis strain 168 (trp-)-competent cells were performed as previously described [33]. QINGSONG was used for the oral administration experiment as a silkworm strain reared and kept in our laboratory. Cloning of Human Growth Hormone Gene The hgh-specific primers, hgh-F: 50 -GGCCATGGTTCCCA ACCATTCCCTTATC-30 (NcoI site is underlined) and hgh-R: 50 -CGAGCTCTTAGAAGCCACAGCTGCCCTC-30 (SacI site is underlined), were designed to amplify an 573-bp hgh gene (GenBank accession no. BC 075012) from the vector pCR4-TOPO. The PCR product was ligated into the pMD18-T vector to prepare plasmid pMD18-hGH. Sequencing of the cloned PCR product was carried out by Sangon Biotech (Shanghai) Co., Ltd., China. Construction of Plasmid pJS700-hGH and Recombinant B. subtilis To obtain an integration of the cotC-hgh fusion gene with an enterokinase site (Asp-Asp-Asp-Asp-Lys) at the B. subtilis amyE, a recombinant plasmid for double crossover with B. subtilis chromosome was constructed. First, plasmid pMD18-hGH and pET30a(?) were digested, respectively, with NcoI and SacI, and purified with Omega BioTek Gel Extraction Kit. Next, the purified hgh gene was inserted into prepared pET30a(?) to construct recombinant plasmid pET30a-hGH, in which between the upstream of the hgh gene and the downstream of Restriction Enzyme cutting site KpnI included an enterokinase site. Finally, we used the restriction endonuclease KpnI and SacI to digest the plasmid pET30a-hGH and pJS700, and inserted the hgh gene into pJS700, resulting in recombinant plasmid pJS700-hGH; in which the upstream and downstream of the region erythromycin (Em)-cotC-hGH was homologous to B. subtilis amyE, as was verified by sequencing [23]. Plasmid pJS700-hGH was digested with BglII, and the resulted linear fragment containing Em-cotC-hGH gene with an enterokinase site (Asp-Asp-Asp-Asp-Lys) was purified with the Gel Extraction Kit and then transformed into the B. subtilis strain 168 (trp-)-competent cells. The
C. Lian et al.: Human Growth Hormone on Bacillus subtilis Spores
transformed cells were incubated in 2 ml SOB medium and cultured at 37 °C overnight with gentle shaking (*80 rpm) for 40 min, followed by spraying onto LB plate containing 0.4 lg/ml erythromycin. Plates were incubated at 37 °C overnight, Emr colonies were selected, and a colony with cotC-hgh integrated at the B. subtilis amyE locus was identified by analysis of amylase activity and then confirmed by PCR. Screening of Mutants with CotC-hGH Integrated at amyE Locus The integration of cotC-hgh at amyE locus will disrupt amyE gene (GenBank accession no. NP_388186.2) causing amylase-negative phenotype on LB plates containing 1 % starch [33]. After incubation at 37 °C overnight, the plates were stained by iodine to examine the amylase activity. A blue color was produced by starch–iodine reaction in the presence of cotC-hgh fusion in the B. subtilis chromosome. Otherwise, no blue color was observed at the B. subtilis 168 (trp-) control when iodine was added, since the expression of amyE resulted in hydrolysis of the starch in the plate. Chromosomal DNA was extracted from both the B. subtilis 168 (trp-) control and cotC-hgh transformants for site-directed PCR. Primers amyE-F: 50 -ATTGCTCGGGCT GTATGACTGG-30 and amyE-R: 50 -GTTACACCATCAC TGTTCGTTCCTT-30 will amplify about 1-kbp wild-type fragment and a 4.3-kb integrated fragment. Primer pair hgh-F and hgh-R was used to detect the correct insertion of the hgh gene. Expression of CotC-hGH on B. subtilis Spore Surface Sporulation of wild-type B. subtilis (trp-) and recombinant strain cotC-hGH was induced in Difco sporulation medium (DSM) by the exhaustion method as previously described [31]. Cultures were harvested 48 h after the initiation of sporulation. Spores were collected, washed several times, and performed by lysosome treatment as described by Nicholson and Setlow to break any residual sporulating cells [31]. Phenylmethylsulfonylfluoride (PMSF, 0.05 M) was included to inhibit proteolysis. Spore coat proteins in suspensions at high density ([1 9 1010 spores/ml) were extracted from B. subtilis (trp-) spores and the cotC-hGH spores using an SDS–DTT extraction buffer as previously described [33]. Western Blot Analysis and Fluorescence Immunoassay Extracted coat proteins were mixed with SDS-PAGE loading buffer and separated on a 12 % polyacrylamide gel at room temperature. Electrophoresis was at 80 V for
30 min and then at 120 V for 150 min. The gel was stained with 0.25 % Coomassie blue R-250 for 1 h and then destained for 12 h using a mixture of 10 % acetic acid and 5 % ethanol. To confirm that cotC-hGH was expressed on the B. subtilis spore surface, the proteins in the gel were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore) for Western blotting using hGH-specific antibody at a dilution of 1:1,000. Immunoreactive proteins were visualized using goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (HRP) as described [13]. In addition, B. subtilis spores cultured in DSM were collected and used for fluorescence immunoassay using hGH-specific antibody and FITC-conjugated goat antirabbit IgG. Fluorescence was examined by excitation at 488 nm with Confocal laser scanning microscope (CLSM, Leica, Germany). Oral Administration of Silkworm with Spores and Hemolymph Protein Extraction for Western Blot Analysis In order to verify the hGH protein that displayed on the B. subtilis spore surface and whether it could be digested and absorbed, the test that the oral administration of silkworm with B. subtilis spores was performed in vivo. Newly molted fifth instar healthy larvae was starved for 6 h, and then inoculated individually through oral administration with 10 ll suspension spores of concentration of 2 9 1010 spores/ml once day [25]. After 5 days normal rearing, the hemolymph in silkworm was collected for protein concentration measurement, which was determined spectrophotometrically at 595 nm using bovine serum albumin for the standard curve determinations [5]. Hemolymph samples (5 larvae, 200 ll hemolymph) were collected from severed third prolegs of fifth instar larvae. The larvae were reared on mulberry leaves in laboratory conditions (25 ± 1 °C, 65 % room humidity and 14-h light:10-h dark). There were 90 larvae averagely split into 3 groups: one control group (C), another wild-type spore treatment group (WT), and the final transgenic spore treatment group (TG). Proteins extracted from silkworms’ hemolymph in each group were fractionated on 12 % denaturing polyacrylamide gels, and then the Western blot method was performed as the same above.
Results Construction of Integrative Vector Containing cotChgh Gene with an Enterokinase Site The strategy to obtain recombinant B. subtilis spores expressing hGH with an enterokinase site on their surface
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was based on the use of the cotC gene and its promoter for the construction of translational fusions and on chromosomal integration of the cotC-hgh gene with an enterokinase site inserting into the coding sequence of the amyE gene. Then the hgh gene with a enterokinase site was obtained from the digestion of plasmid pET-30a-hGH with the restriction endonuclease KpnI and SacI, and cloned at the 30 -end of cotC open reading frame in plasmid pJS700 to generate the recombinant plasmid pJS700-hGH, in which hGH and cotC were co-expressed under the control of cotC promoter (Fig. 1a). The obtained fusion gene was integrated into the B. subtilis 168 chromosome at the amyE locus by a double crossover event (Fig. 1b). Identification of cotC-hpi Integrated in B. subtilis Chromosome The recombinant B. subtilis having integrated cotC-hgh into chromosome at the amyE locus by double crossover was first analyzed by amylase activity. The integration of cotC-hgh at amyE locus interrupted the expression of amylase. As a result, no transparent halo was observed around the recombinant clones on a starch-containing plate stained by iodine potassium iodide (Fig. 2a). It was indicated that the recombinant gene cotC-hgh was successfully inserted into B. subtilis chromosome. But the wild-type B. subtilis produced a transparent halo around the clones due to the expression of amylase (Fig. 2a). Disruption of amyE in B. subtilis chromosome and correct insertion of cotChgh at the amyE locus were further confirmed by PCR with four primer pairs. Primer pair amyE-F/amyE-R amplified a 1,098-bp (amylase gene) and 3,800-bp (fusion gene) fragment from wild-type B. subtilis (WT) and transgenic B. subtilis (TG) chromosome, respectively. Moreover, primer pairs hgh-F/hgh-R, amyE-F/hgh-R, and hgh-F/amyE-R produced fragments around 1.8 kb, 4.3 kb, and 2.3 kb on the transgenic B. subtilis chromosome, respectively. However, no PCR product was observed in wild-type B. subtilis chromosome (Fig. 2b). Display of hGH on Recombinant Spores To confirm that hGH was successfully expressed on recombinant B. subtilis spores, hGH-specific antibody was used to perform Western blot analysis. The result showed that a 30.8 kDa clear band was detected in the extraction from recombinant spores (Fig. 3, Lane 2). Since the part of cotC gene of B. subtilis encodes a 66 amino acid protein with a predicted molecular mass of 8.8 kDa [8], and the ORF of hgh encodes a 191 amino acid protein with a molecular mass of 22 kDa, the Western blot analysis suggested that the recombinant fusion protein was expressed with expected molecular mass. However, no similar band
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Fig. 1 Cloning strategy. a The flow chart of plasmid pJS700-hGH c construction. amyE 50 -end and amyE 30 -end are integrative fragments from amylase gene of B. subtilis 168 (trp-); Emr, erythromycinresistant site; cotC, a B. subtilis spore coat protein-encoding gene. b The strategy for the chromosomal integration of the cotC-hGH gene fusions. Arrows indicate the positions of primer pairs used in the confirmation of the hgh gene insertion in the mutant strain and wild type
in the extraction proteins from wild-type B. subtilis spores was detected (Fig. 3, Lane 1). Therefore, the result indicated that fusion protein cotC-hGH was expressed in the spore correctly. In order to verify that hGH was displayed on the spore surface, fluorescence immunoassay was performed with rabbit anti-hGH primary antibodies and FITC-conjugated goat anti-rabbit secondary antibodies. Bright green fluorescence could be observed on the surface of recombinant spores (Fig. 4) with CLSM, but no fluorescence signal was observed on the surface of wild-type B. subtilis spores (Control). This demonstrated that hGH was expressed on the surface of recombinant spores. Oral Administration of Silkworm with Spores Groups of silkworm were inoculated by the oral administration with recombinant spores of hGH-expressing strain, isogenic control strain and double-distilled water. In order to confirm that the hGH protein displayed on the recombinant spores could be digested and absorbed into the hemolymph of silkworm for the addition of enterokinase site (Asp-Asp-Asp-Asp-Lys), proteins extracted from silkworms’ hemolymph in each group were fractionated on 12 % denaturing polyacrylamide gels, and then the Western blot method was performed using previously prepared hGH-specific antibody. As shown in Fig. 5, a 22-kDa band was detected in the extracts from the hemolymph of TGtreated silkworm, while no similar band in the WT-treated group was detected, indicating that the hGH protein displayed on the recombinant spores could be digested and absorbed into the hemolymph of silkworm. In addition, this also demonstrated that the hgh gene was successfully displayed on the surface of B. subtilis spores.
Discussion The bacterial surface display technology has been attracting a great deal of attention due to their applications in the fields of vaccine, drug delivery vehicles, whole-cell biocatalysts, biosensors, environmental remediation, and biomolecule screening platforms [22]. Among various microbial cell surface display systems presently available,
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the newly developed spore surface display offers additional advantages such as higher stability and safety. Compared to other Bacillus species such as Bacillus anthracis [27] and Bacillus thuringiensis [32], B. subtilis has more advantages in developing spore surface display system due to the detailed knowledge of its spore structure [12]. B. subtilis spore display system is based on the construction of gene fusion between heterologous DNA and a B. subtilis gene coding for a component of the spore coat, where the spore coat protein has been shown to be located on its outside surface [12, 15]. So far three different spore coat proteins namely cotB, cotC, and cotG have been used [15, 34]. Among the three proteins, the content of cotC is up to 50 % [21], which means it should be a ideal fusion partner for expressing heterologous proteins. In this paper, we displayed hGH on the B. subtilis spore surface by fusion hgh to the C-terminal of cotC. This improvement may be contributed by the resistant characters of B. subtilis spores. It was reported that B. subtilis spores could survive in Fig. 2 Identification of the transgenic B. subtilis. a Identification of transgenic B. subtilis by amylase activity analysis. 1 Transgenic strains and wild-type B. subtilis growing on LB plate containing 1 % starch. 2 The plate stained with iodine. The insertion of cotC-hgh destroyed amyE and made the strain deficient in amylase, while the wild-type strain showed a big white halo around clones due to the secretion of amylase. b PCR analysis with different primer pairs. M, 250-bp DNA ladder marker; WT wild-type B. subtilis; TG transgenic B. subtilis with cotC-hgh insertion; primer pairs used in PCR are labeled above
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gastric environment, reach the intestinal tract, and germinate to vegetative cells in vivo. Results obtained from PCR, Western blot, amylase activity analysis, and immunofluorescence microscopy demonstrated that the cotC-hGH fusion protein was correctly exposed on the spore surface. Noteworthily, we found that the protein hGH we got was solube; therefore, the product should have a competitive edge on the future market. The study reported here is the first to describe hGH displayed on the surface of spores so far. To evaluate the effect of an enterokinase site in the fusion protein cotC-hGH, we used silkworms as a model. Immunoblot analysis of hemolymph in silkworm by oral administration with B. subtilis spores implied that the hGH protein displayed on the spores surface may be digested and absorbed into the silkworm hemolymph. This change may be contributed by the addition of enterokinase site, the resistant properties of B. subtilis spore, and the simple intestinal structure of silkworm.
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Generally, the use of mammals for drug development is expensive and highly problematic with regard to ethical issues. Currently, the similarities in the pharmacokinetics of drug between silkworms and mammals, including the mechanisms of chemical absorption, distribution, metabolism, and excretion have been widely reported [10, 11, 17, 18]. Moreover, the silkworm body size is large enough for hemolymph preparations and organ isolation, which are essential for studying the pharmacodynamics of drugs in animal bodies. In addition, it is far less costly to rear silkworms than mammals, and a large number of larvae can
Fig. 3 Western blot analysis of proteins extracted from B. subtilis spores. Fusion protein cotC-hGH was detected by hGH-specific antibody. Lane 1 wild-type B. subtilis spore proteins. Lane 2 recombinant B. subtilis spore proteins
be maintained in a small space. Screening of therapeutic agents can be easily performed with a large number of individual silkworms without the same ethical concerns involved in the use of mammals. Thus, we will further choose the use of silkworms as model animals for evaluating the therapeutic effects of cotC-hGH.
Fig. 5 Western blot analysis of proteins extracted from hemolymph in silkworm after oral administration with B. subtilis spores. Lane 1 hemolymph proteins in silkworm oral administrated with recombinant B. subtilis spores; lane 2 hemolymph proteins in silkworm oral administrated with wild-type spores; and lane 3 hemolymph proteins in silkworm oral administrated with ddH2O
Fig. 4 Immunofluorescent detection of recombinant spores. Bright-field images and immunofluorescent images of wild-type (control) and recombinant B. subtilis spores (10 9 100 oil immersion lens)
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Taken together, this study has displayed hGH on B. subtilis spores, and oral administration in silkworm suggested that the hGH protein displayed on the recombinant spores may be absorbed into its hemolymph, which laid a perfect foundation for evaluating the effect of cotC-hGH in the near future. Acknowledgments We honestly thank Professor Degang Ning for kindly gifting plasmid pJS700, and Professor Hengchuan Xia for language revision of the text. This study was supported by the National Basic Research Program of China ‘‘973’’ under Grant No. 2012CB114604, the Natural Science Research General Project of Education Office of Anhui Province (No. KJ2012B108), and Postgraduate Research and Innovation Project of Jiangsu Province (Nos. CXZZ12_0703 and CXZZ13_0699). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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