Porcine Growth Hormone Research Articles

Characterization of a Truncated Form of Recombinant Porcine Growth Hormone Generated in Vitro During Solubilization of Inclusion Bodies

During the development of a novel solubilization procedure (1) for bacterial inclusion bodies (IB′s) using the cationic surfactant cetyltrimethylammonium chloride (CTAC; (CH 3) 3N +C 16H 33Cl) significant proportions of an apparently truncated, lower molecular weight (MW) variant form of recombinant pig growth hormone (rPGH) were observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis relative to pig pituitary derived GH. The formation of this rPGH-like species, designated P-band, was found to occur in vitro during solubilization of IB′s by CTAC and was dependent on pH and temperature of solubilization, but was not due directly to the use of CTAC, as purified soluble rPGH of the correct MW could not be converted to P-band by exposure to CTAC alone. The bacterial proteolysis suspected as being responsible for the in vitro formation of P-band could not be inhibited by the use of a "cocktail" of defined antiproteolytic agents but was inhibited by pH and temperature, and by solubilization of IB′s in 5% SDS, 6 M gnHCl or 7.5 M urea. Detailed characterization of the structure of P-band by N-terminal amino acid sequencing, electrospray mass spectrometry, radio-receptor binding assay, peptide mapping, and C-terminal peptide sequencing confirmed that P-band was approximately 950 mass units smaller than normal rPGH and lacked eight C-terminal amino acids. A significant finding was that P-band is unable to bind to the pig liver-membrane GH receptor in a competitive radioreceptor assay. Analysis of the relative secondary and tertiary structure of P-band by circular dichroism spectra, intrinsic tryptophan-dependent fluorescence, and average surface hydrophobicity (2) suggested small but measurable changes to the overall structure of P-band relative to normal rPGH. Consequently, our results also suggest that the C-terminal portion of rPGH, including in particular the last eight amino acids, is of major importance in the binding of rPGH to the pig liver membrane GH receptor.

Development of an enzyme immunoassay for the determination of porcine growth hormone in plasma

A competitive enzyme immunoassay for the determination of porcine growth hormone (pGH) was developed. The assay is based on (anti-rabbit IgG) sheep IgG-coated microtitre plates, anti-pGH from rabbits, biotinyl-pGH and streptavidin-horseradish peroxidase. The assay is done directly with 40 μl of plasma and the calibration graph (90% relative binding at 0.36 ng ml −1 and 50% relative binding at 4.4 ng ml −1) is prepared in plasma without measurable endogenous pGH. Recovery of pGH added to different plasma samples amounted to 102–119%, and all variabilities were < 8.6%. The assay shows 50% cross-reaction with recombinant pGH, but no significant binding of porcine prolactin, follicle stimulating hormone and luteinizing hormone. The assay detects high and low plasma pGH levels within the physiological variation as well as changes in plasma pGH after stimulation with growth hormone releasing factor or treatment with recombinant pGH. Hence it offers a reliable alternative to radioimmunoassay. However, a reference method providing evidence on the identity of exogenous pGH will be required for proving illegal treatment with pGH and for forensic purposes.

Characterization of a bicistronic retroviral vector composed of the swine vesicular disease virus internal ribosome entry site.

We cloned the 5' nontranslated region (NTR) from the genome of swine vesicular disease virus (SVDV), a member of the family Picornaviridae, and used it to construct a bicistronic retroviral vector. The vector is characterized by coexpression of two genes from a single transcript. We found that inclusion of the 5' NTR of SVDV did not negate the viral vector titer. Protein analysis indicated that the 5' NTR could efficiently direct internal initiation, thus allowing the downstream gene to be translated. Translation of the internally initiated porcine growth hormone gene was about 30-fold less than that when the porcine growth hormone gene was at the upstream position in NIH 3T3 cells but was about equivalent to that in HeLa cells, implying that some cellular factors that stimulated internal initiation of the SVDV 5' NTR are present in HeLa cells. However, in G418-selected clones, the Neor-encoding gene was expressed with equivalent efficiency either at a downstream position or at an upstream position in either NIH 3T3 or HeLa cells. Compared with the conventional double-gene vector or the U3-based vector, the bicistronic vector coexpressed two genes much more efficiently, owing to elimination of promoter interference. Furthermore, this type of vector infected and expressed the target genes efficiently in two primary cell lines, rat embryo and human skin fibroblast cells, which we tested. These experimental data suggest a better design for the retroviral vector and provide evidence that internal initiation of the SVDV 5' NTR was stimulated cell specifically.

Effect of Murine Ascites on the Ability of Hybridoma Cells to Produce Antibody and Proliferate In Vitro

Murine ascites has been shown to contain a variety of growth promoting activities. In this study, we examined the effects of ascites fluid on the efficiency of hybridoma production and cell growth. SP2/0 mouse myeloma cells were injected into peritoneal cavities of mice and ascitic fluid was collected. Lymphocytes from mice immunized with porcine growth hormone (pGH) were fused with myeloma cells in a standard hybridization procedure. These cells were then dispensed in 96-well plates in medium containing either 2.5% ascites or 20% fetal calf serum (FCS) and cultured for few days. Supernatants from these cultures were collected and analyzed for anti-pGH antibodies. It was demonstrated that ascites-supplemented medium increased the efficiency in generating specific antibody-secreting hybridomas by 4-fold over FCS-supplemented medium. Furthermore, hybridoma cells were cultured in microtiter plate and found to proliferate in response to ascites in a dose dependent manner. This effect was abolished by prior digestion of ascites with trypsin, indicating its protein nature. B-lymphocyte related cytokines seemed less likely involved because antibodies to IL-4 and IL-6 failed to alter the stimulatory effect of ascites. Ascites was fractionated by FPLC using Superose 12 column and the active moiety was found to be a small m.w. peptide (< 1,000 dalton). Therefore, murine ascites is capable of substituting for conventional FCS in culture medium in the area of hybridoma technology.

Apal and Cfo I polymorphisms in the porcine growth hormone gene

<i>Apal</i> and <i>Cfo</i> I polymorphisms in the porcine growth hormone gene

Growth hormone response to GRF and insulin-induced hypoglycemia in Yorkshire and Meishan pigs.

Plasma glucose and porcine growth hormone (GH) responses to insulin and/or growth hormone-releasing factor (GRF) challenges were determined in Yorkshire (Y) and Meishan (MS) castrated male pigs at 140 days of age. The decline in glucose levels observed during the first 30 min after insulin injection was similar between the two breeds. In Y pigs, GH concentrations increased sharply after insulin injection. The GH response to insulin was much smaller in MS than in Y pigs (101 +/- 82 vs. 1,077 +/- 124 ng.min.ml-1, P < 0.001 after 0.30 U/kg insulin). The GH response to GRF (10 micrograms/kg) did not differ significantly between the two breeds. The GH response was larger after combined insulin (0.20 U/kg) plus GRF than after GRF alone in Y pigs (1,512 +/- 229 vs. 463 +/- 401 ng.min.ml-1, P < 0.05), whereas it did not differ in MS pigs (714 +/- 164 vs. 507 +/- 127 ng.min.ml-1, P > 0.10). The present data demonstrate that the GH response to insulin is dramatically altered in MS pigs. A possible explanation to this result is that somatostatin tone would not be depressed by insulin-induced hypoglycemia in MS pigs.

Molecular Biological Studies on Animal Growth Hormones

AbstractThe pituitary growth hormone (GH) from various vertebrate species could be purified from crude pituitary extracts by a single‐step HPLC using a reverse‐phase C18 column. The same procedure was also useful for peptide mapping of proteins. Pharmacokinetic analysis of porcine GH revealed that it circulated in plasma with a half‐life of 2 h and that most injected GH was confined to blood and probably lymph, with a total body clearance of porcine GH around 8 ± 3 mL h−1 kg−1. Several GH have been sequenced by using cloning of their cDNA. The comparative analysis of these cDNA reveals the conserved structural characteristics for all vertebrate GH: 1. There are 2 disulfide bonds in the single polypeptide chain of the GH molecule which are supposedly necessary to maintain the native conformation of the hormone. 2. The invariant residues are predominantly located within the α‐helices which are also necessary to maintain the structural integrity but may not be required for species specificity. 3. The putative signal peptides of various GH deduced from the cDNA sequences show the general consensus pattern of hydrophobicity but exhibit no obvious sequence homology. These characteristics are in agreement with other signal peptides of most secretory proteins. A high‐level expression of porcine GH in E. coli was achieved by using some combination of promoter and transcriptional terminator. The expressed 22‐kDa GH was immunoreactive to the specific GH antibody and showed expected partial N‐terminal amino‐acid sequence.

Application of an optimized immunostaining technique to evaluate the heterogeneous secretory response from porcine somatotropes by cell blotting.

The objective of the present study was to quantify the absolute hormone release from individual porcine pituitary cells incubated on polyvinylidene difluoride (PVDF) transfer membranes (cell-blot assay). After immunoperoxidase staining, growth hormone (GH) release from isolated somatotrope cells appeared like a colored zone of secretion surrounding the cell. Optical densities of these secretion zones were quantitated by computerized image analysis and translated into picograms by means of an appropriate standard curve. As a prior step, the staining method and the optimal immunocytochemical conditions were selected by applying purified porcine growth hormone (pGH) to the transfer membranes. The avidin-biotin-peroxidase nickel-intensified (ABC-Ni) method produced a better resolution than the peroxide-anti-peroxidase (PAP) method, although both techniques were similar with regard to background, sensitivity, and range of quantitation. The amount of GH released from single porcine somatotropes was highly heterogeneous, although the cells were treated under the same conditions. Moreover, this fact was consistent with the stimulation of the average release of GH by GH-releasing factor (GHRF) but not by GHRF+somatostatin (SRIF). Our results confirm the availability of the recently developed cell-blot assay and support the concept of functional heterogeneity in anterior pituitary cell populations.

Effects of growth hormone and triiodothyronine on insulin-induced progesterone production by granulosa cells from prepubertal gilts

Two experiments were performed using granulosa cells from medium-sized follicles (2–4 mm) derived from prepubertal gilts. Cells were cultured in a serum-free medium at a density of either 1 or 2 × 106 viable cells per well (experiments 1 and 2, respectively). For exp. 1, porcine growth hormone (pGH) (0 or 100 ng mL−1) was included in the culture medium from the time of plating, and low-density lipoprotein (LDL) (100 μg mL−1) was added at 72 h. For exp. 2, granulosa cells were plated in a culture medium containing either pGH (0 or 100 ng mL−1) or triiodothyronine (T3) (0 or 5 ng mL−1) or both pGH T3; LDL was not included. For both experiments, after 24 h of culture, bovine insulin at 0, 10, 100 or 1000 ng mL−1 was included in the medium. Hormones were replaced at 48 and 72 h, and the cultures were terminated at 96 h. Results from exp. 1 indicated that insulin increased (P &lt; 0.01) progesterone production in a dose-dependent manner, both in the presence and absence of LDL. This response was augmented (P &lt; 0.01) by co-culture with pGH. Results from exp. 2 confirmed the augmenting effect of pGH (P &lt; 0.01). It was further observed that T3 increased (P &lt; 0.01) progesterone production when cultured with insulin at 1000 ng mL−1, but at lower insulin-inclusion levels, results were equivocal. The progesterone production response was greatest (P &lt; 0.01) when cells were cultured with both pGH and T3 at insulin levels of 100 or 1000 ng mL−1. There appeared to be little relationship between the media concentrations of insulin-like growth factor 1 and progesterone. The present results suggest that relatively high levels of pGH and T3 will enhance the in vitro steroidogenic capabilities of porcine granulosa cells. Key words: Granulosa cells, GH, T3, insulin

Immunodominant structures of human growth hormone identified by homolog-scanning mutagenesis

Homolog-scanning mutagenesis has been reported to be useful in elucidating the antigenic epitopes recognized by monoclonal antibodies and hGH binding to its receptor. However, little is known about which structures are recognized as immunodominant by murine serum antibodies. Therefore, the previously published series of hGH homologs and additional mutants of human placental lactogen (hPL), porcine growth hormone (pGH), and human prolactin (hPRL) were examined for their interaction with murine serum derived anti-hGH antibodies. As compared to wild-type hGH, nine of the nineteen segment substituted mutants tested showed a significant reduction in binding to anti-hGH sera. These disruptive substitutions mapped to 5 regions on a structural model of hGH: the length of helix 1 (residues 11–33), the loop between the first disulfide bond and helix 2 (residues 54–74), the beginning of helix 3 (residues 109–112), the carboxyl half of helix 4 (residues 167–182), and the final carboxyl terminus segment of the molecule (residues 184–191). In terms of the current structural model, three of the five immunodominant regions (the loop between residues 54–74, central portion of helix 4 to the carboxyl terminus and part of the amino terminus region of helix 1) closely overlaps the hGH receptor binding epitopes.

Equilibrium denaturation of recombinant porcine growth hormone.

Equilibrium denaturation of recombinant porcine growth hormone (pGH) derived from Escherichia coli using the denaturant guanidine hydrochloride (GuHCl) was followed by ultraviolet absorption spectroscopy, intrinsic fluorescence, far-ultraviolet circular dichroism, and size-exclusion chromatography. The normalized denaturation transition curves for each of the above methods were not coincident; denaturation resulted in an initial disruption of the tertiary structure, whereas secondary structure and degree of compactness were disrupted at higher concentrations of denaturant. Size-exclusion chromatography also detected an associated form of pGH at intermediate GuHCl concentrations. These findings conclusively show that pGH does not follow a simple two-state folding mechanism but are consistent with the framework model of folding. Stable intermediates observed were similar to those seen in other nonhuman growth hormones and are characterized as compact and largely alpha-helical yet lacking nativelike tertiary structure.

A relationship between the starting secondary structure of recombinant porcine growth hormone solubilised from inclusion bodies and the yield of native (monomeric) protein after in vitro refolding

Recombinant porcine growth hormone (rPGH) was solubilised from inclusion bodies (IB's) using either 6 M guanidinium hydrochloride (GnHCl), 7.5 M urca or by a novel method using a cationic surfactant, cetyltrimethylammonium chloride (CTAC). Circular dichroism (CD) analysis of the secondary (2°) structure of the urea- and GnHCl-solubilised rPGH showed the absence of α-helical content with the majority of the molecule existing in a ‘random coil’ structure. In contrast, the CTAC-solubilised rPGH displayed significant starting 2° structure (10–15% α helix; 30–40% β structure). The three rPGH preparations were refolded in vitro against weak urea, GnHCl or aqueous buffers, resulting in an average refolding efficiency of 50% native (monomeric) rPGH for CTAC solubilised IB's and only 20% for urea or GnHCl solubilised IB's. We conclude that the method of solubilisation of IB's and the resultant difference in the starting 2° structure of rPGH, particularly α-helical content, is a major in vitro factor that apparently predetermines the aggregation/refolding behaviour rPGH irrespective of refolding environment.

Novel recombinant fusion protein analogues of insulin-like growth factor (IGF)-I indicate the relative importance of IGF-binding protein and receptor binding for enhanced biological potency.

An efficient expression system in Escherichia coli for several biologically active insulin-like growth factor-I (IGF-I) fusion peptide analogues is described. These novel IGF-I fusion protein analogues have properties that make them very useful reagents in the investigation of IGF-I action. The analogues comprise an IGF-I sequence and the first 11 amino acids of methionyl porcine growth hormone (pGH) and include [Met1]-pGH(1-11)-Val-Asn-IGF-I, which contains the authentic IGF-I sequence, and two analogues, [Met1]-pGH(1-11)-Val-Asn-[Gly3]-IGF-I and [Met1]-pGH(1-11)-Val-Asn-[Arg3]-IGF-I, where Glu-3 in the human IGF-I sequence has been replaced by Gly or Arg respectively. The three peptides are referred to as Long IGF-I, Long [Gly3]-IGF-I or Long [Arg3]-IGF-I depending on the IGF-I sequence present. Production of the purified fusion peptides was aided by folding the reduced and denatured fusion peptide sequence under conditions that gave very high yields of biologically active product. Introduction of a hydrophobic N-terminal extension peptide appears to facilitate the correct folding of the IGF-I analogues compared with that obtained previously when folding normal-length IGFs. The biological activities of the IGF-I fusion peptides were compared with authentic IGF-I and the truncated analogue, des(1-3)IGF-I. In L6 rat myoblasts, all the analogues were more potent than authentic IGF-I in their abilities to stimulate protein and DNA synthesis and inhibit protein breakdown. In H35 hepatoma cells, where the IGFs act through the insulin receptor, the Long IGF-I analogues maintained a similar potency relative to IGF-I as was observed in the L6 myoblasts. The order of biological potency in cell lines secreting IGF-binding proteins (IGFBPs) into the medium was Long [Arg3]-IGF-I-des(1-3)IGF-I greater than Long [Gly3]-IGF-I greater than Long IGF-I greater than IGF-I. In chicken embryo fibroblasts, a cell line that does not secrete detectable IGFBPs into the medium, Long [Arg3]-IGF-I, was less potent than IGF-I. Investigation of receptor and IGFBP association by these analogues reinforced our previous findings that N-terminal analogues of IGF-I show increased biological potency due to changes in the degree of their IGFBP interactions.

Effect of frequency of administration of exogenous porcine growth hormone on growth and carcass traits and ovarian function of prepubertal gilts.

Three experiments were conducted to examine relationships among dose and frequency of administration of exogenous porcine growth hormone (pGH) on growth traits and ovarian function of prepubertal gilts. In Exp. 1, gilts were treated with 0 or 5 mg of pGH daily for 42 d or 5 mg of pGH daily on alternate weeks over a 42-d period. In Exp. 2, gilts were treated with 0, 2.5, or 5 mg of pGH daily for 31 d or daily on alternate weeks for 31 d. In Exp. 3, gilts received 5 mg of pGH daily on either wk 1, 3, and 5 or wk 2, 4, and 6 during a 42-d period. In all experiments, ADG increased dramatically and feed efficiency improved markedly during treatment with pGH, and both traits declined rapidly during periods when treatment was withdrawn. Gilts treated with pGH daily on alternate weeks tended to be more similar (P greater than .05) to control gilts for growth rate, feed efficiency, and carcass measurements than to gilts that received continuous daily administration of pGH during the entire duration of the experiments. Increased concentrations of estradiol and insulin-like growth factor (IGF)-I in follicular fluid and serum, decreased concentrations of IGF-II in follicular fluid, and increased weights of ovaries were evident as both dose and frequency of exogenous pGH administration increased. Therefore, gilts are extremely sensitive to administration and withdrawal of exogenous pGH during the finishing phase of the production cycle and can respond within 7 d to changes in exogenous pGH treatment regimens. Alternate weekly administration of exogenous pGH in vivo may improve follicular function, as indicated by relationships among IGF-I and IGF-II, estradiol, and progesterone, but fails to improve overall growth and carcass traits compared with controls.

Effects of exogenous porcine growth hormone (GH) on bone metabolism in young pigs

Effects of exogenous porcine growth hormone (GH) on bone metabolism in young pigs

HaeII and MspI polymorphisms are detected in the second intron of the porcine growth hormone gene.

HaeII and MspI polymorphisms are detected in the second intron of the porcine growth hormone gene.

High-density Escherichia coli cultivation process for hyperexpression of recombinant porcine growth hormone

Fermentation studies were performed on an Escherichia coli culture that carries a recombinant plasmid composed of an ampicillin-resistant gene, a temperature-regulated pL promoter, and a porcine pituitary cDNA sequence coding for growth hormone. The objective was to achieve high cell density while maintaining the specific expression level of recombinant porcine growth hormone (r-pGH) observed in shake flasks. At a specific expression level of 20% of total cell protein, the cell density of a glucose-limited fed-batch process reached 38 units of OD 600 in 14 h, compared to flask cultivation, which resulted in only 1.4 units of OD 600 in the same period. The observed critical fermentation conditions for maximal expression included (1) limiting glucose concentration below 1 g l -1 throughout the fed-batch growth and induction phases, (2) keeping postinduction temperature at 42°C for 5–7 h, and (3) maintaining a postinduction growth rate around 0.17–0.21 h -1.

A proposed mechanism of action of a growth hormone-specific monoclonal antibody in the enhancement of hormonal activity

The potentiation of the biological activity of recombinant porcine growth hormone (pGH) by immunologic manipulation was investigated. An anti-pGH monoclonal antibody, designated PS-7.6, was generated and its effect on pGH was evaluated in hypophysectomized (hypox) rats. As expected, administration with pGH for 5 consecutive days promoted these animals to grow. The effect was further augmented when pGH was given together with PS-7.6 antibody and the enhancing ability of the antibody lasted beyond the treatment period. The growth profile of rats receiving antibody alone did not differ from that of untreated controls, indicating that PS-7.6 antibody by itself was not a growth stimulant. The possible mechanism of action of the antibody was investigated by analyzing blood and tissue samples of animals following injection with 125I-labeled pGH either in its free form or complexed with PS-7.6 antibody. As compared to the pGH levels in animals receiving free pGH, approximately a half pGH was released into circulation from the injection sites when it was given in a complex form. Furthermore, 2–4-fold increases in pGH deposition were also found in various tissues of animals treated with pGH-antibody complexes over that of respective tissues of animals receiving free pGH. Therefore, the present findings suggest that PS-7.6 antibody is capable of augmenting the somatogenesis of pGH and the effect is, at least in part, explainable by its ability in altering the pharmacokinetics and biodistribution of pGH in animals.

The interrelationship between and the regulation of hepatic growth hormone receptors and circulating GH binding protein in the pig

We evaluated the interrelationship between, and regulation of, the hepatic growth hormone receptor and serum GH binding protein (GH BP) in pigs treated with recombinant porcine growth hormone (rpGH). Infant and pubertal male pigs (N = 5 per group) received either rpGH 0.15 mg/kg daily or diluent intramuscularly for 12 days. Somatic growth, serum IGF-I and GH BP and [125I]bovine GH (bGH) binding to MgCl2-treated hepatic membrane homogenates were examined. Marked age-related increases were seen in serum GH BP (p less than 0.001) and [125I]bGH binding to hepatic membranes (p less than 0.001). GH BP was increased in rpGH treated animals (p = 0.03), from 13.8 +/- 1.2 (mean +/- 1 x SEM) (controls) to 17.8 +/- 2.0% in infants, and from 35.2 +/- 2.6 (controls) to 41.8 +/- 3.4% in pubertal animals. [125I]bGH binding to hepatic membranes was also increased by rpGH treatment (p less than 0.05), from 7.0 +/- 1.6 (controls) to 15.4 +/- 3.6% in infants and from 53.7 +/- 7.1 (controls) to 65.1 +/- 11.8% in pubertal animals. No significant interaction between age and treatment was seen. Overall, serum GH BP correlated significantly with [125I]bGH membrane capacity (r = 0.82, p less than 0.001), with a correlation of r = 0.83 in the infant animals but no significant correlation in the pubertal animals considered alone (r = 0.13). Serum IGF-I correlated significantly with serum GH BP (r = 0.93, p less than 0.001) and [125I]bGH membrane binding capacity (r = 0.91, p less than 0.001). These observations suggest that serum GH BP levels reflect major changes of hepatic GH receptor status.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of hormonal activity with a monoclonal antibody specific to porcine growth hormone

Abstract The potentiation of the biological effects of recombinant porcine growth hormone (pGH) by immunologic manipulation was investigated. A monoclonal antibody (mAb), designated PS‐7.6, was raised against pGH and repeatedly shown to enhance the responsiveness of hypophysectomized (hypox) rats to pGH. As a result, animals receiving a combination treatment of pGH and mAb PS‐7.6 together gained significantly more weight than those receiving the same doses of pGH alone. The enhancing action of the mAb was a rapid process and its effective doses ranged from 0.1 to 2 mg/injection. The ability of the antibody to augment the hormonal activity persisted beyond the 5‐day treatment period and the differences in net weight gain between treated and control animals remained significant for 28 days. Results from treatment frequency studies further suggested that pGH when complexed with mAb PS‐7.6 required fewer injections and produced a greater efficacy than being administered alone. Therefore, present findings suggest that mAb PS‐7.6 may prove useful for not only improving the efficiency of pGH, but also developing a novel formulation for sustained pGH release.