Nb2 Cells Research Articles

Design, development and evaluation of PEGylated rhGH with preserving its bioactivity at highest level after modification

Modification of recombinant proteins with polyethylene-glycol (PEG) can improve their pharmacokinetic properties, although their bioactivity may be reduced after PEGylation due to structural changes. In this study, simultaneous optimization of PEGylation efficiency and preserved bioactivity of recombinant human growth hormone (rhGH) was investigated. In this regard, experiments were designed by the response surface methodology (RSM)-central composite design (CCD) utilizing design expert software. Under the obtained optimum conditions of 6.73 molar ratio of PEG to protein and pH 7.71 as the main factors affect the process, 54% PEGylation efficiency and 63% preserved bioactivity can be achieved. Based on the ANOVA table, model F-values equal to 31.16 and 20.8 for PEGylation efficiency and preserved bioactivity, respectively, demonstrated the validity and importance of the models. High performance liquid chromatography (HPLC) and gel electrophorese analyses verified the purity of the PEGylated form of rhGH. Findings showed that the modified protein would be stable for six months at 4 °C. In vitro cell growth assessments revealed Nb2-11 cell proliferation during 48 h, although proliferation rate decrease with the increase of PEGylated rhGH concentration. Half-life prolongation in serum observed for PEGylated form in comparison with the non-modified one on in vivo. In overall, the results are promising for the utilization of the PEGylated form of rhGH for the treatment of human growth deficiency after further investigations.

High efficient prokaryotic expression and purification of bioactive human growth hormone using a cleavable self-aggregating tag

Human growth hormone (hGH) is synthesized by the anterior pituitary gland and promotes cell proliferation and growth. This protein has been authorized to use for the treatment of various human growth disorders and until recently, substantial efforts have been made to upgrade the previous introduced strategies. Due to the small size of hGH and absence of posttranslational modifications, Escherichia coli is the ideal host for hGH production. In the present work, we employed a previously established cleavable self-aggregating tag (cSAT) for the expression and purification of hGH in BL21 (DE3) strain of E. coli to evaluate its effectiveness. The tag is composed of a self-cleavable intein and a self-assembling peptide ELK16 (Mxe GyrA intein-ELK16). At the first step, an active insoluble aggregate of the recombinant hGH-Mxe GyrA intein-ELK16 fusion protein was expressed through an efficient T7 based expression system and then purified with a simple centrifugation. The thiol reagent dithiothreitol (DTT) was then added to induce the intein-mediated cleavage and as a result the peptide released into the soluble fraction. Afterward, the hGH production was determined by SDS-PAGE and then the final concentration of released hGH was measured by the Bradford assay (4.96 mg/ml). Furthermore, the bioactivity of purified hGHs was confirmed by calculating its growth-stimulating effect using Nb2 cell line proliferation assay. All in all, the current study offers a straightforward and fast procedure for the production of pure and bioactive hGH in E. coli.

A 60 kDa prolactin variant secreted by cervical cancer cells modulates apoptosis and cytokine production.

Prolactin (PRL) is associated with different types of cancer, such as cervical cancer. Recombinant PRL has antiapoptotic effect on cervical cancer cells, and it can also induce cytokine production on macrophages. A 60kDa variant of PRL is produced by cervical cancer cells. The aim of the present study was to evaluate this variant's bioactivity, to test its effect on cervical cancer cell apoptosis, and to assess its ability to induce cytokine production on THP-1 macrophages. First, 60kDa PRL was isolated and used to stimulate Nb2 cells. Later, apoptosis was measured after exposure to 60kDa PRL. Finally, cytokines were measured on THP-1 stimulated supernatants. Our results show that 60kDa PRL increased Nb2 cell proliferation. Apoptosis was decreased after stimuli with 60kDa PRL in cervical cancer cells. IL-1β and TNF-α are produced by THP-1 macrophages after stimuli. These results suggest that 60kDa PRL produced by cervical cancer cells is able to reduce apoptosis in HeLa, SiHa and C-33A cells and induce IL-1β and TNF-α production by THP-1 macrophages.

Sustained Release of Recombinant Human Growth Hormone from Bioresorbable Poly(ester urea) Nanofibers

Recombinant human growth hormone (rhGH) has been used clinically for several decades. However, rhGH has a short half-life in vivo and therefore for efficacy requires frequent subcutaneous injection, leading to pain and poor patient compliance. An effective rhGH delivery system able to continuously release rhGH over a comparatively long period (generally over a month) would be a significant clinical advance. In this study, a novel long-duration release strategy of rhGH was developed by encapsulating sugar glass-stabilized rhGH in electrospun, bioresorbable poly(ester urea) (PEU) nanofibers. The rhGH was found to be randomly dispersed among the fibers, and sustained rhGH release with only a modest burst was observed for at least 6 weeks as studied by a bicinchoninic acid (BCA) protein assay. Significantly, the released rhGH remained bioavailable and bioactive as determined by an Nb2 cell bioassay specific for rhGH. Our results suggest the feasibility of this system as an effective long-term sustained releas...

Internal image anti-idiotypic antibody: A new strategy for the development a new category of prolactin receptor (PRLR) antagonist

Over the past decades, a number of prolactin receptor (PRLR) antagonists have been developed, which can be divided into two categories, PRLR analogue and anti-PRLR antibody. However, until now, there have been no commercially available PRLR antagonists. Here, we described a new approach for the preparation of PRLR antagonist, namely internal image anti-idiotypic antibody strategy. The hybridoma technique was used to generate anti-idiotypic antibodies to PRL. Competitive ELISA, competitive receptor-binding analysis and immunofluorescence assay (IFA) were then used to screen and characterize anti-idiotypic antibodies to PRL. One internal image anti-idiotypic antibody, termed MG7, was obtained. A series of experiments demonstrated that MG7 behaved as a typical internal image anti-idiotypic antibody (Ab2β). MG7 exhibited effective antagonistic activity, which not only inhibited PRL binding to PRLR in a dose-dependent manner but also inhibited PRLR-mediated intracellular signalling. Furthermore, MG7 also blocked Nb2 cell proliferation induced by PRL. The current study suggests that MG7 has the potential application in the PRL/PRLR-related studies in future. In addition, this work also suggests that the internal image anti-idiotypic antibody may represent a novel strategy for the development of PRLR antagonist.

Development of a Novel, Anti-idiotypic Monoclonal Anti-prolactin Antibody That Mimics the Physiological Functions of Prolactin.

In this work, we prepared a panel of monoclonal anti-idiotypic antibodies to ovine prolactin (oPRL) by the hybridoma technique. Among these antibodies, one anti-idotypic antibody (designated B7) was chosen for further characterization by a series of experiments. We first demonstrated that B7 behaved as a typical Ab2β based on a series of enzyme-linked immunosorbent assays. Subsequently, the results of a competitive receptor-binding assay confirmed that B7 could specifically bind to the prolactin receptor (PRLR) expressed on target cells. Finally, we examined its biological activities in CHO-PRLR and Nb2 cells and observed that B7 could activate Janus kinase 2-signal transducer and activator of transcription signalling in CHO-PRLR and Nb2 cells and induce BaF3 proliferation. The present study suggests that i) B7 can serve as a PRLR agonist or PRL mimic and has potential applications in regulating mammary gland development, milk production and maintenance of lactation in domestic animals and ii) B7 may be a biological reagent that can be used to explore the mechanism of PRLR-mediated intracellular signalling.

Preclinical characterization of eleven new Cys-PEGylated hGH mutants

We synthesized and tested for biological activity eleven new PEGylated hGH derivatives. To this aim we used different strategies. A first group of PEGylable Cys derivatives was prepared by mutating into Cys single specific aminoacid residues located either in the connecting loop between helices 1 and 2 (Ile36 and Phe44) or in the connecting loop between helices 3 and 4 (Ile138 and Phe146). A second group of mutants was synthetized by inserting new Cys close to the aforementioned positions. Other PEGylable mutants were prepared by inserting a Gly4–Cys chain either at the N- or at the C-terminus of the hGH molecule. Finally, a Cys residue of the second hGH disulfide bond (Cys182–Cys189) was made available for PEGylation by mutating its companion Cys to open up the disulfide bridge. All these mutants were tested for their ability to affect Nb2 cell proliferation in vitro and showed different effects. Two mutations (Phe44Cys and +Cys47) severely impaired and two (Ile138Cys and Phe146Cys) increased hGH bioactivity. The mutation Cys189Ser was functionally silent whereas the remaining mutations caused a 15–50% decrease in activity. 20kDa-pegylation caused a dramatic decrease in bioactivity in all mutants. The r-hGH-(Ile138Cys)–Cys138–PEG derivative showed the highest activity (15% of wild-type unpegylated hGH) and was selected for pharmacockinetic studies in vivo. It showed a fivefold longer half-life and was significantly more effective than wild-type hGH in causing weight gain when given subcutaneously twice a week to hypophysectomized rats.In conclusion, even when it is directed to residues supposed to have a marginal role in the activity of this hormone, 20kDa pegylation has detrimental effect on hGH bioactivity. These effects may be counterbalanced by the increase in half-life as it happens in pegylated Ile138Cys–hGH that could represent a promising new long-acting hGH derivative.Focal points:•Bedside: Current therapy of GH deficiency still has the important limitation of being delivered by daily subcutaneous injections and this reduces the compliance of the small pediatric patients. There is, therefore interest in developing long-acting GH derivative such as the new ones that we present here.•Benchside: By investigating the effect of Cys substitutions at aminoacidic positions that had not been investigated in previous studies, we obtained evidence that Ile138 and Phe146 have a role in GH bioactivity in vitro.•Industry: The mono-pegylated hGH mutant described in this work represents a promising candidate for human clinical pharmacology studies as long-acting derivative of h-GH.•Community: By decreasing the frequency of GH injections, long acting GH could decrease the costs of GH treatment and increase medication adherence.

Expression of autocrine prolactin and the short isoform of prolactin receptor are associated with inflammatory response and apoptosis in monocytes stimulated with Mycobacterium bovis proteins

Increased levels of prolactin (PRL) have recently been associated with carcinogenesis and the exacerbation of autoimmune diseases, and might be involved in the progression of tuberculosis (TB). To investigate the relationship between PRL and prolactin receptor (PRLr) expression with inflammatory response and apoptosis in monocytes, we used THP-1 cells stimulated with antigens of the Mycobacterium bovis AN5 strain culture filtrate protein (CFP-M. bovis). Western blot (WB), real-time Polymerase chain reaction (PCR), and immunocytochemistry were performed to identify both PRL and PRLr molecules. PRL bioactivity and proinflammatory cytokine detection were assessed. The results showed that PRL and PRLr messenger RNA (mRNA) were synthesized in THP-1 monocytes induced with CFP-M. bovis at peaks of 176- and 404-fold, respectively. PRL forms of 60 and 80kDa and PRLr isoforms of 40, 50, and 65kDa were also identified as time-dependent, while 60-kDa PRL, as well as 40-, and 50-kDa PRLr, were found as soluble forms in culture media and later in the nucleus of THP-1 monocytes. PRL of 60kDa released by monocytes exhibited bioactivity in Nb2 cells, and both synthesized PRL and synthesized PRLr were related with nitrite and proinflammatory cytokine levels proapoptotic activity in CFP-M. bovis-induced monocytes. Our results suggest the overexpression of a full-autocrine loop of PRL and PRLr in monocytes that enhances the inflammatory response and apoptosis after priming with M. bovis antigens.

High-level expression and purification of recombinant human growth hormone produced in soluble form in Escherichia coli

Human growth hormone (hGH) was one of the first recombinant proteins approved for the treatment of human growth disorders. Its small size (191 amino acids), possession of only 2 disulphide bonds and absence of posttranslational modifications make Escherichia coli the host of choice for its production on any scale. In this work, we have utilized an efficient T7 based expression system to produce high levels of soluble thioredoxin-hGH (Trx-hGH) fusion protein. We outline a relatively simple three step purification process employing two immobilized metal-affinity chromatography and one anion-exchange steps and removal of fusion partner by enterokinase cleavage yielding native hGH. The ability of cell populations to produce quantities of up to 1g/L of the soluble Trx-hGH fusion protein has been tested in flask cultivations as well as in batch and fed-batch bioreactor runs. The sequence and structure of derived hGH were confirmed by mass spectrometry and circular dichroism and its native function, to induce cell proliferation, was confirmed by employing a Nb2 cell line proliferation assay.

Prokaryotic Soluble Overexpression and Purification of Bioactive Human Growth Hormone by Fusion to Thioredoxin, Maltose Binding Protein, and Protein Disulfide Isomerase

Human growth hormone (hGH) is synthesized by somatotroph cells of the anterior pituitary gland and induces cell proliferation and growth. This protein has been approved for the treatment of various conditions, including hGH deficiency, chronic renal failure, and Turner syndrome. Efficient production of hGH in Escherichia coli (E. coli) has proven difficult because the E. coli-expressed hormone tends to aggregate and form inclusion bodies, resulting in poor solubility. In this study, seven N-terminal fusion partners, hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A (NusA), protein disulfide bond isomerase (PDI), and the b′a′ domain of PDI (PDIb′a′), were tested for soluble overexpression of codon-optimized hGH in E. coli. We found that MBP and hPDI tags significantly increased the solubility of the hormone. In addition, lowering the expression temperature to 18°C also dramatically increased the solubility of all the fusion proteins. We purified hGH from MBP-, PDIb′a′-, or Trx-tagged hGH expressed at 18°C in E. coli using simple chromatographic techniques and compared the final purity, yield, and activity of hGH to assess the impact of each partner protein. Purified hGH was highly pure on silver-stained gel and contained very low levels of endotoxin. On average, ∼37 mg, ∼12 mg, and ∼7 mg of hGH were obtained from 500 mL-cell cultures of Trx-hGH, MBP-hGH, and PDIb′a′-hGH, respectively. Subsequently, hGH was analyzed using mass spectroscopy to confirm the presence of two intra-molecular disulfide bonds. The bioactivity of purified hGHs was demonstrated using Nb2-11 cell.

Use of frequency swept ultrasound to manipulate Nb2-11 live cells in a microfluidic channel

Recently microfluidic manipulation of live cell has been attracting attention because it is necessary for fast and accurate bioassays of cells within miniaturized devices like a lab-on-a-chip. This work concerns the alignment, translocation and concentration of live cells in a microfluidic channel by utilizing an ultrasonic standing wave. Among many ultrasonic manipulation methods, the technique using frequency swept ultrasound which was proposed in a previous study offers excellent particle manipulation performance, but that has been demonstrated only for biocompatible polystyrene microspheres with several different diameters, not actual biological cells. However, the technique needs to be verified for real cells because sizes and properties are little different with each other. Thus, in this work, the feasibility of using the ultrasonic frequency sweep method to manipulate live Nb2-11 cells was investigated experimentally The cells were aligned in a plane, moved to one side of a channel, and finally filtered out by sending them to one outlet with ultrasound. The frequency region of the applied ultrasound was between 1.75 MHz and 3.05 MHz. From experimental results, the cell behavior showing perfect alignment, almost perfect translocation and hence considerable aggregation proved the feasibility of using the frequency sweep method though the force acting on the Nb2-11 cells are much smaller than the force acting on polystyrene beads due to the differences in their sizes and mechanical properties.

Reevaluation of the proposed autocrine proliferative function of prolactin in breast cancer

The pituitary hormone prolactin (PRL) has been implicated in tumourigenesis. Expression of PRL and its receptor (PRLR) was reported in human breast epithelium and breast cancer cells. It was suggested that PRL may act as an autocrine/paracrine growth factor. Here, we addressed the role of locally synthesised PRL in breast cancer. We analysed the expression of PRL in human breast cancer tumours using qPCR analysis and in situ hybridization (ISH). PRL mRNA expression was very low or undetectable in the majority of samples in three cDNA arrays representing samples from 144 breast cancer patients and in 13 of 14 breast cancer cell lines when analysed by qPCR. In accordance, PRL expression did not reach detectable levels in any of the 19 human breast carcinomas or 5 cell lines, which were analysed using a validated ISH protocol. Two T47D-derived breast cancer cell lines were stably transfected with PRL-expressing constructs. Conditioned medium from the T47D/PRL clones promoted proliferation of lactogen-dependent Nb2 cells and control T47D cells. Surprisingly, the PRL-producing clones themselves displayed a lower proliferation rate as compared to the control cells. Their PRLR protein level was reduced and the cells were no longer responsive to exogenous recombinant PRL. Taken together, these data strongly indicate that autocrine PRL signalling is unlikely to be a general mechanism promoting tumour growth in breast cancer patients.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-013-2731-7) contains supplementary material, which is available to authorized users.

Lipopolysaccharide induces the expression of an autocrine prolactin loop enhancing inflammatory response in monocytes

BackgroundProlactin from pituitary gland helps maintain homeostasis but it is also released in immune cells where its function is not completely understood. Pleiotropic functions of prolactin (PRL) might be mediated by different isoforms of its receptor (PRLr).MethodsThe aim of this study was to investigate the relationship between the eventual synthesis of PRL and PRLr isoforms with the inflammatory response in monocytes. We used THP-1 and monocytes isolated from healthy subjects stimulated with lipopolysaccharide (LPS). Western blot, real time PCR and immunocytochemistry were performed to identify both molecules. The bioactivity of the PRL was assessed using a bioassay and ELISA to detect pro inflammatory cytokines.ResultsPRLr mRNA and PRL mRNA were synthesized in THP-1 monocytes activated with LPS with peaks of 300-fold and 130-fold, respectively. The long (100 kDa) and the intermediate (50 kDa) isoforms of PRLr and big PRL (60 kDa) were time-dependent upregulated for monocytes stimulated with LPS. This expression was confirmed in monocytes from healthy subjects. The PRLr intermediate isoform and the big PRL were found soluble in the culture media and later in the nucleus in THP-1 monocytes stimulated with LPS. Big PRL released by monocytes showed bioactivity in Nb2 Cells, and both PRL and PRLr, synthesized by monocytes were related with levels of nitrites and proinflammatory citokines.ConclusionsOur results suggest the expression of a full-autocrine loop of PRL enhances the inflammatory response in activated monocytes. This response mediated by big PRL may contribute to the eradication of potential pathogens during innate immune response in monocytes but may also contribute to inflammatory disorders.

Periplasmic production via the pET expression system of soluble, bioactive human growth hormone

A pET based expression system for the production of recombinant human growth hormone (hGH) directed to the Escherichia coli periplasmic space was developed. The pET22b plasmid was used as a template for creating vectors that encode hGH fused to either a pelB or ompA secretion signal under control of the strong bacteriophage T7 promoter. The pelB- and ompA-hGH constructs expressed in BL21 (λDE3)-RIPL E. coli are secreted into the periplasm which facilitates isolation of soluble hGH by selective disruption of the outer membrane. A carboxy-terminal poly-histidine tag enabled purification by Ni2+ affinity chromatography with an average yield of 1.4mg/L culture of purified hGH, independent of secretion signal. Purified pelB- and ompA-hGH are monomeric based on size exclusion chromatography with an intact mass corresponding to mature hGH indicating proper cleavage of the signal peptide and folding in the periplasm. Both pelB- and ompA-hGH bind the hGH receptor with high affinity and potently stimulate Nb2 cell growth. These results demonstrate that the pET expression system is suitable for the rapid and simple isolation of bioactive, soluble hGH from E. coli.

Expression of human growth hormone in the milk of transgenic rabbits with transgene mapped to the telomere region of chromosome 7q

The advent of transgenic technology has provided methods for the production of pharmaceuticals by the isolation of these proteins from transgenic animals. The mammary gland has been focused on as a bioreactor, since milk is easily collected from lactating animals and protein production can be expressed at very high levels, including hormones and enzymes. We demonstrate here the expression pattern of recombinant human growth hormone (rhGH) in transgenic rabbits carrying hGH genomic sequences driven by the rat whey acidic protein (WAP) promoter. The transgene was mapped to the q26-27 telomere region of chromosome 7q by fluorescence in situ hybridization (FISH). Nearly 30 % of the F1 generation demonstrated the presence of transgene. The recombinant growth hormone was detected in the milk of the transgenic rabbit females, but not in serum, up to the level of 10 μg/ml. Ectopic expression of the transgene in the brain, heart, kidney, liver, and salivary gland was not observed, indicating that a short sequence of rat WAP promoter (969 bp) contained essential sequences directing expression exclusively to the mammary gland. The biological activity of recombinant growth hormone was measured by immunoreactivity and the capability to stimulate growth of the hormone-dependent Nb211 cell line.

Preparation and characterization of recombinant human growth hormone–Zn2+-dextran nanoparticles using aqueous phase–aqueous phase emulsion

An effective and simple method was developed to prepare spherical and uniform-sized recombinant human growth hormone (rhGH)–Zn2+-dextran nanoparticles (NPs) for a variety of delivery applications of the delicate protein. The results showed that the prepared rhGH-Zn2+-dextran particles were spherical, glassy, and uniform in size. rhGH recovered from the NPs maintained its structural integrity and its bioactivity well, as suggested by the Nb2-11 cell proliferation assay. Using polyethylene glycol and polysaccharide to mediate Zn2+-induced precipitation has proved to be a promising method to prepare spherical protein-loaded NPs with desired sizes and preserved protein stability. From the Clinical EditorThis basic science paper discusses a simple yet effective method to prepare spherical and uniform-sized recombinant human growth hormone (rhGH)-Zn2+-dextran nanoparticles for a variety of delivery applications.

Editorial: Is macroprolactinemia just a diagnostic pitfall?

As hyperprolactinemia is involved in the impairment of the gonadotropic axis, serum prolactin (PRL) assessment is often required in the clinical scenario of menstrual disturbances, infertility, loss of libido, galactorrhea, osteoporosis, and bone fractures [1–3]. Nevertheless, the excessive requirement of PRL determination led to an important laboratory pitfall: the find of hyperprolactinemia in subjects with few or no symptoms related to such condition. Human PRL in circulation has marked size heterogeneity, mainly with three forms: 23 kDa (monomeric or little PRL), 50 kDa (dimeric or big PRL), and 150–170 kDa (big–big prolactin or macroprolactin, the three forms being indistinguishable by routine assays [4]. Nevertheless, monomeric PRL, the most common form, is considered to be the biological active one, whereas molecular aggregates as macroprolactin are regarded to have low biologic activity. Therefore, the predominance of macroprolactin, associated with normal levels of monomeric PRL, may lead to the laboratorial diagnosis of hyperprolactinemia in subjects with no symptoms, ending up in unnecessary treatment approaches. The gel-filtration chromatography is the goldstandard method to confirm the presence of big–big PRL, but is a costly and time-consuming process. For routine purposes, precipitation with polyethylene glycol (PEG) is an excellent screening method [5]. The predominant molecular form recovered (i.e., assayed after precipitation) is the highly biologically active monomeric PRL. Usually considering macroprolactin [60% of total PRL, macroprolactinemia has been shown as a common finding, occurring in 8–42% of all cases of hyperprolactinemia. The reason for such large range can be attributed both to selection bias and the use of diverse assays, which differently recognize the amount of big–big PRL in each sample [6]. Macroprolactin biologic activity is still controversial in the literature. Studies in vitro with rat Nb2 cell bioassays show either its presence or absence. To explain the presence of activity in vitro but not in vivo, it was hypothesized that its high molecular weight impairs capillary barrier crossing and its action on target cells. Moreover, the Nb2 cells are a PRL-dependent rat immune cell line expressing a mutant form of PRL receptor, so a bioassay using cells harboring human PRL receptors which addresses the biologic activity of macroprolactin should be more reliable. In fact, a study by Glezer et al. [7] showed that sera of individuals with macroprolactinemia presented lower biological activity in a bioassay using a mouse cell transfected with the long form of human PRL receptor as compared to the rat Nb2 bioassay. Moreover, Hattori et al. [8] assume that the bioactivity of macroprolactin in the Nb2 bioassay is due to dissociation of monomeric PRL from the autoantibodies as a result of the longer incubation than in a bioassay using human breast cancer cells. Despite these controversies in the literature concerning the biologic activity of PRL aggregates, most patients with macroprolactinemia do not manifest clinical features related to hyperprolactinemia. This Endocrine issue brings two publications from Turkey addressing the macroprolactin matter. In one study, Isik et al. [9] enrolled 337 hyperprolactinemic individuals, and found that in 26.1% of them macroprolactinemia prevailed, based on a percentage of PRL in the serum supernatant \40% after PEG precipitation. Subjects with PRL recover value of C40% were considered with absence of M. D. Bronstein (&) Neuroendocrine Unit, Division of Endocrinology and Metabolism, Hospital das Clinicas, University of Sao Paulo, Sao Paulo, SP, Brazil e-mail: mdbronstein@uol.com.br

Growth hormone secretory pattern in non-obese children and adolescents with Prader-Willi syndrome

The aetiology of impaired growth hormone (GH) secretion in Prader-Willi syndrome (PWS) remains controversial due to the common occurrence of obesity. To further clarify whether suboptimal GH secretion in PWS is an artefact of excess weight, we evaluated both GH immunological activity and GH bioactivity after arginine administration in 23 non-obese PWS patients [seven females, aged 6.9 +/- 0.9 years, body mass index (BMI) SDS 0.63 +/- 0.26], in comparison with a control group of 32 healthy subjects, matched for age, gender and BMI (10 females, aged 7.9 +/- 0.3 years, BMI SDS 0.21 +/- 0.20). Serum GH concentration was measured with a time-resolved immunofluorometric assay (IFMA), while GH bioactivity was evaluated by the Nb2 cell bioassay. Serum IGF-I concentrations were measured by double-antibody RIA. GH mean peak after pharmacological stimulation was significantly lower in PWS individuals compared with controls when measured either by IFMA (6.05 +/- 1.23 microg/L vs. 23.7 +/- 1.06 microg/L, p < 0.0001) or by Nb2 (6.87 +/- 0.55 microg/L vs. 12.88 +/- 0.19 microg/L, p < 0.0001). Analysis of integrated GH secretion (AUC) confirmed that the PWS group differed significantly from the control subjects (387.9 +/- 76.1 microg/L/h vs. 1498.1 +/- 56.2 microg/L/h, p < 0.0001); the same result was obtained when the GH rise after arginine administration was expressed as nAUC (278.2 +/- 53.3 microg/L/h vs. 1443.6 +/- 52.5 microg/L/h, p < 0.0001). PWS patients had an IGF-I SDS significantly lower than those found in control subjects (p < 0.0001). Subnormal IGF-I values were present in 19 PWS individuals (82.6%) and two healthy controls (6.2%). These findings are in agreement with the hypothesis that a complex derangement of hypothalamus-pituitary axis occurs in PWS.

Human growth hormone–transferrin fusion protein for oral delivery in hypophysectomized rats

Transferrin (Tf)-based recombinant fusion protein approach was investigated to achieve oral delivery for human growth hormone (hGH). Plasmid constructs expressing the fusion proteins were established by fusing coding sequences of both hGH and Tf in frame. Fusion proteins were produced in serum free media by transient transfection of human embryonic kidney HEK293 cells. The SDS-PAGE analysis of conditioned media showed that fusion proteins expressed at high purity with a 100 kDa molecular weight; the Western blot analysis with anti-hGH and anti-Tf antibodies verified the identity of fusion proteins. The Nb2 cell proliferation and Caco-2 cell Tf receptor (TfR) binding assays demonstrated that fusion proteins retained bioactivity of both hGH and Tf, respectively. A helical linker was inserted as spacer between hGH- and Tf-domain to enhance the bioactivity and the yield of the fusion protein. Two fusion proteins, hGH-Tf (GT) and hGH-(H4) 2-Tf (GHT) were obtained and assessed in hGH-deficient hypophysectomized rats for in vivo biological activity. Results from seven-day subcutaneous dosing (1.25 mg/kg/day) demonstrated that both GT and GHT fusion proteins were bioactive in vivo, comparable to native hGH. However, only the GHT, but not GT, fusion protein promoted a modest but statistically significant weight gain after oral dosing with 12.5 mg/kg/day.

High throughput purification of recombinant human growth hormone using radial flow chromatography

Recombinant human growth hormone (r-hGH) was expressed in Escherichia coli as inclusion bodies. Using fed-batch fermentation process, around 670 mg/L of r-hGH was produced at a cell OD600 of 35. Cell lysis followed by detergent washing resulted in semi-purified inclusion bodies with more than 80% purity. Purified inclusion bodies were homogenous in preparation having an average size of 0.6 μm. Inclusion bodies were solubilized at pH 12 in presence of 2 M urea and refolded by pulsatile dilution. Refolded protein was purified with DEAE-anion exchange chromatography using both radial and axial flow column (50 ml bed volume each). Higher buffer flow rate (30 ml/min) in radial flow column helped in reducing the batch processing time for purification of refolded r-hGH. Radial column based purification resulted in high throughput recovery of diluted refolded r-hGH in comparison to axial column. More than 40% of inclusion body protein could be refolded into bioactive form using the above method in a single batch. Purified r-hGH was analyzed by mass spectroscopy and found to be bioactive by Nb2 cell line proliferation assay. Inclusion body enrichment, mild solubilization, pulsatile refolding and radial flow chromatography worked co-operatively to improve the overall recovery of bioactive protein from inclusion bodies.