Optimization of the solubilization and renaturation of fish growth hormone produced by Escherichia coli

Growth hormone (GH) enhances the growth rate of aquacultured fish and shellfish, but it is difficult to extract native GH from fish pituitary glands. However, fish recombinant GH (rGH) can be efficiently synthesized by Escherichia coli cells, although it exists in denatured form in inclusion bodies (IB). We studied the solubilization of IB and the renaturation of rGH to help facilitate the production of a large amount of biologically active rGH. A 100-ml sample of rGH-producing E. coli produced 73.43 ± 5.47 mg IB (dry weight, n = 3) after 20 h induction by 1 mM isopropyl β-o-thiogalactopyranoside. Interestingly, if the bacteria were induced by 0.1 mM β-lactose, 95.3 ± 3.43 mg of IB was obtained. The optimal conditions for denaturation and renaturation of rGH were when IB were solubilized in 6 M guanidine hydrochloride and then dialysed against pH 10 dialysis buffer (50 mM ammonium bicarbonate and 2 mM EDTA) containing 100 mM l-arginine, 2 mM oxidized glutathione and 2 mM reduced glutathione for 24 h at 4 °C in a volume ratio of 3 to 500. At least 20% of the denaturated rGH in IB was renatured. Juvenile black sea bream injected with 0.05 μg/g resultant rGH once every 2 weeks exhibited significant increases (P < 0.05) in weight gain (84%) relative to fish in the control group over a 16-week period. This process is an economical and effective way to obtain an active form of rGH biosynthesized by a prokaryotic system. Received: 18 November 1996 / Received revision: 5 March 1997 / Accepted: 7 March 1997     

Expression and characterization of the Neurospora crassa endoglucanase GH5-1

Filamentous fungi secrete a wide range of enzymes, including cellulases and hemicellulases, with potential applications in the production of lignocellulosic biofuels. Of the cellulolytic fungi, Hypocrea jecorina (anamorph Trichoderma reesei) is the best characterized in terms of cellulose degradation, but other cellulolytic fungi, such as the model filamentous fungus Neurospora crassa, can serve a crucial role in building our knowledge about the fungal response to biomass due to the many molecular and genetic tools available for this organism. Here we cloned and expressed GH5-1 (NCU00762), a secreted endoglucanase in N. crassa. The protein was produced using a ccg-1 promoter under conditions in which no other cellulases are present. Native GH5-1 (nGH5-1) and this recombinant GH5-1 (rGH5-1) were purified to gauge differences in glycosylation and activity; both rGH5-1 and nGH5-1 were similarly glycosylated, with an estimated molecular weight of 52 kDa. On azo-carboxymethylcellulose, rGH5-1 activity was equal to that of nGH5-1, and on cellulose (Avicel) rGH5-1 was 20% more active. The activity of a GH5-1-GFP fusion protein (rGH5-1-GFP-6xHis) was similar to rGH5-1 and nGH5-1. To determine the binding pattern of catalytically active rGH5-1-GFP-6xHis to plant cell walls, Arabidopsis seedlings were incubated with rGH5-1-GFP-6xHis or Pontamine Fast Scarlet 4B (S4B), a cellulose-specific dye. Confocal microscopy showed that rGH5-1-GFP-6xHis bound in linear, longitudinal patterns on seedling roots, similar to S4B. The functional expression and characterization of rGH5-1 and its GFP fusion derivative set important precedents for further investigation of biomass degradation by filamentous fungi, especially N. crassa, with applications for characterization and manipulation of novel enzymes.     

Escherichia coli-produced fish growth hormone as a feed additive to enhance the growth of juvenile black seabream (Acanthopagrus schlegeli)

Recombinant growth hormone (rGH) of fish was highly efficiently synthesized by Escherichia coli cells harbouring an expression plasmid (pKLYP) containing a 600 base pairs segment of cDNA which encoded the mature region of yellowfin porgy ( Acanthopagrus latus ) pre-growth hormone. After dena-turation and renaturation, rGH purified from inclusion bodies became the refolding form. rGH were then processed into the fishmeal pellet at various concentrations. Juvenile black sea-bream ( Acanthopagrus schlegeli ) fed twice daily with meal containing 0.5% rGH exhibited significant increases (P < 0.01) in percentage weight gain (60%) and feed efficiency (41%) relative to fish in the control group at week 12. Growth enhancement was maintained for at least 4 weeks (at week 16) after the termination of treatment.     

Induction of growth hormone release by glycyrrhizae radix on rat

Induction of growth hormone (GH) by Glycyrrhizae Radix (GR), one of the most popular herbal medicine, and its major ingredients were studied in rat pituitary cells in vitro and in vivo assay. The MeOH extract and the n-hexane (HX) fraction of GR induced rat GH (rGH) release up to 1.89 times (0.34 +/- 0.04 nM) and 4.59 times (0.83 +/- 0.03 nM), compared to the basal level (p < 0.05). Among many ingredients isolated and purified from GR both glycyrrhetinic acid and glycyrrhizin induced significantly rGH release compared to the control (p < 0.05). After an intravenous injection of rat growth hormone releasing hormone (rGHRH) (10 microg/kg) as positive control, in SD rats, Tmax of plasma rGH level was 10 min, C(max) was 3.84 +/- 0.01 nM (n = 3), and enhanced plasma rGH level returned to the baseline in 90 min. Both AUC(0-90) (area under the curve) of plasma rGH level after HX fraction and that after rGHRH administration were increased significantly from the basal level, respectively (p < 0.01). In conclusions, HX fraction is the most active fraction of MeOH extract of GR in rGH induction.     

Induction of growth hormone release by dioscin from Dioscorea batatas DECNE

In this study, dioscin was isolated from Dioscoreae Rhizoma (DR), which is the rhizome of Dioscorea batatas D(ECNE). that inhabits broad areas of Korea and Japan. To determine whether dioscin induced growth hormone (GH) release, we evaluated its induction effects on GH release both in vitro and in vivo. The 70% methanol extract of DR, and its n-hexane and n-BuOH fractions, induced rat GH (rGH) release in rat pituitary cells 10-fold, 8-fold, and 5- fold higher than the control (0.36 +/- 0.02 nM), respectively (p < 0.05 each). The dioscin-induced rGH release of the cells was concentration-dependent and its ED(50) was 1.14 x 10(-5) M. Within 90 minutes after intravenous administration of 10 microg/kg (p < 0.05 at t(max)), dioscin caused the greatest increase in rGH concentration (C(max)) in the rat plasma (34.16 +/- 14.10 ng/ml) (n = 4), which was twice as high as the control group (12.88 +/- 3.29 ng/ml) (n = 27).     

The effect of recombinant GH treatment on ovarian growth and atresia in sheep

This study investigated the effect of recombinant bovine GH (rGH) on follicle development and LH secretion patterns in ewes. In Experiment 1, 20 ewes (n=10/group) synchronized with progestagen sponges on Day 0 received either a 7 d period of rGH treatment starting on Day 4, or acted as controls. On Day 11, all ewes were unilaterally ovariectomized. Follicles in the excised ovary were characterized on the basis of size, health status and rate of granulosa cell proliferation. Circulating levels of LH, GH, IGF-1 and insulin were monitored. Compared to controls, rGH treatment significantly increased the number of healthy follicles >2.0 mm, reduced the number of 0.25 to 0.5-mm follicles and reduced the number of 0.8 to 2.0-mm early atretic follicles. GH treatment also reduced the mitotic index of 0.25 to 0.5-mm follicles. Recombinant GH treatment had no effect on LH secretion patterns, but plasma GH, IGF-1 and insulin levels were increased in rGH-treated ewes. Because rGH treatment may have had an anti-atresia effect in Experiment 1, the hypothesis for Experiment 2 was that rGH treatment could maintain follicle development beyond 2.5-mm diameter in bovine follicular fluid (bFF)-treated ewes. Forty ewes (n=10/group) were synchronized with progestagen sponges. Starting 5 d after sponge insertion, ewes were treated for 6 d with rGH, bFF, rGH plus bFF, or acted as controls. On Day 12, ewes were sacrificed, and follicles were dissected out of their ovaries and assessed on the basis of size. FSH concentrations were assessed on Days 7, 9 and 11. GH treatment alone significantly increased the number of 2.5 to 4.0-mm follicles compared to controls, whereas no follicles larger than 2.5 mm were present in bFF-treated ewes. In rGH plus bFF-treated ewes, the number of 2.5 to 4.0-mm follicles was similar to controls, but there were less follicles >4.0 mm. GH treatment had no effect on FSH levels, whereas bFF treatment significantly reduced FSH levels. These results expand previous findings that rGH treatment of ewes alters follicle development, but do not suggest that rGH treatment is likely to be of benefit in superovulatory protocols. Furthermore, the data indicate that rGH has an anti-atretic action that is unlikely to be mediated via gonadotropins.     

Variations in prolactin and growth hormone production during cellular growth in clonal strains of rat pituitary cells.

A permanent, clonal strain of rat pituitary tumor cells (GH3-cells) spontaneously synthesizes and secretes prolactin (rPRL) and growth hormone (rGH) into the culture medium. The rates of hormone production (microng extracellular hormone/mg cell protein/24 hours) and synthesis (vida infra) as well as the rate of [3H]thymidine incorporation into DNA (DNA synthesis) have been studied. During logarithmic growth rPRL and rGH production increased to 160 and 250% of the value at day 2 after plating, while during the plateau phase of cell growth hormone production decreased to initial values. The fluctuations in rPRL production could be fully explained by variations in the rate of rPRL synthesis: [3H]eucine incorporated into rPRL as measured with immunoprecipitation and polyacryl-amide gel electrophoresis. Also the rates of synthesis and production of rGH showed parallel changes during exponential and plateau phase of growth, but this hormone was probably degraded intracellularly. The relative reduction in the rate of synthesis of rPRL and rGH during the plateau of growth corresponded closely to the fall in the rate of DNA synthesis. The reduction in rPRL synthesis could not be explained through an inhibition by extra-cellular rPRL accumulation or by cell to cell interaction occurring in dense cultures. The intracellular concentrations of both hormones were unaltered during logarithmic growth, but rose to 500% for rPRL and 200% for rGH during the plateau phase. In spite of the marked variations in basal rPRL and rGH production the GH3 cultures of different ages were equally able to increase rPRL and decrease rGH production in response to thyrotropin releasing hormone (3 X 10(-7) M) and 17beta-estradiol (10(-8)M).     

Multihormonal regulation of the human, rat, and bovine growth hormone promoters: differential effects of 3',5'-cyclic adenosine monophosphate, thyroid hormone, and glucocorticoids

We have analyzed the effects of a variety of hormones on activity of the rat GH (rGH), human GH, (hGH), and bovine GH (bGH) promoters. After transient transfection of rat pituitary tumor cells, all three promoters are induced by addition of 8-bromo-cAMP. Sequences required for the cAMP responsiveness of the hGH and rGH promoter lie within 183 base pairs of the mRNA start site. Although the rGH promoter is thyroid hormone (T3) responsive in this system, a construct containing 2.7 kilobases of the hGH promoter 5'-flanking sequences is not. Since we also found that the bGH promoter is T3 responsive in these cells, the hGH results are not likely to be due to a species specific factor required for induction in rat pituitary cells. The hGH promoter is weakly induced by dexamethasone whereas the rGH promoter does not respond to glucocorticoids. The hGH and rGH promoters are not responsive to TRH. These results illustrate the potential heterogeneity in hormonal responses of the same gene in different species.     

<Emphasis Type="Italic">Paenibacillus</Emphasis> sp. A59 GH10 and GH11 Extracellular Endoxylanases: Application in Biomass Bioconversion

The cost-efficient degradation of xylan to fermentable sugars is of particular interest in second generation bioethanol production, feed, food, and pulp and paper industries. Multiple potentially secreted enzymes involved in polysaccharide deconstruction are encoded in the genome of Paenibacillus sp. A59, a xylanolytic soil bacterium, such as three endoxylanases, seven GH43 β-xylosidases, and two GH30 glucuronoxylanases. In secretome analysis of xylan cultures, ten glycoside hydrolases were identified, including the three predicted endoxylanases, confirming their active role. The two uni-modular xylanases, a 32-KDa GH10 and a 20-KDa GH11, were recombinantly expressed and their activity on xylan was confirmed (106 and 85 IU/mg, respectively), with differences in their activity pattern. Both endoxylanases released mainly xylobiose (X2) and xylotriose (X3) from xylan and pre-treated biomasses (wheat straw, barley straw, and sweet corn cob), although only rGH10XynA released xylose (X1). rGH10XynA presented optimal conditions at pH 6, with thermal stability at 45–50 °C, while rGH11XynB showed activity in a wider range of pH, from 5 to 9, and was thermostable only at 45 °C. Moreover, GH11XynB presented sigmoidal kinetics on xylan, indicating possible cooperative binding, which was further supported by the structural model. This study provides a detailed analysis of the complete set of carbohydrate-active enzymes encoded in Paenibacillus sp. A59 genome and those effectively implicated in hemicellulose hydrolysis, contributing to understanding the mechanisms necessary for the bioconversion of this polysaccharide. Moreover, the two main free secreted xylanases, rGH10XynA and rGH11XynB, were fully characterized, supporting their potential application in industrial bioprocesses on lignocellulosic biomass.     

Induction of growth hormone release by Pueraria thunbergiana BENTH.

Puerariae Radix (PR), Puerariae Flos (PF), and Puerariae Surculus (PS) as well as their constituents were tested for induction of rat growth hormone (rGH) release by both rat pituitary cell culture and in vivo experimentation in order to develop them to novel drugs. Through a calibration curve of the rGH released by addition of rat growth hormone-releasing hormone (rGHRH) to rat pituitary cells, the 70 % ethanol extracts of PR and PS increased rGH release by about 1.6 and 1.7 times as high, respectively, as the control group (264.6 +/- 13.6 pM). However, each puerarin type as a representative constituent of PR in Korea Pharmacopeia (KP) and tectorigenin and an important ingredient of PF were twice as effective as in the control group. The acid hydrolysate of Puerariae Surculus (HPS) increased rGH release concentration-dependently, and its EC (50) was approximately 10.4 microg/ml. The T (max) value for rGH after injection of 20 microg/kg of rGHRH was 10 - 30 min, while the C (max) value was increased by approximately 12-fold compared to the control group (198.2 +/- 25.0 pM) and the AUC (0 - 45) was increased to 10 times the level of the control group (10,840.9 +/- 845.5 min. pM). On the other hand, T (max) for the HPS was 60 min, while C (max) was increased approximately to 5.8 fold compared to control (244.1 +/- 36.4 pM). C (max) for puerarin was 1,028.6 +/- 502.7 pM, that is, approximately 5.2 times as high as the control level. However, tectorigenin (20 microg/kg) was of no statistical significance. Therefore, we suggest that the HPS and puerarin act either on GH secretagogue receptors or on GHRH receptor of somatotrophin as possible agonists or an inhibitor on somatostatin receptor to release rGH, respectively.     

The effects of 5-azacytidine on the expression of the rat growth hormone gene. Methylation modulates but does not control growth hormone gene activity

The role of DNA methylation in the expression of the rat growth hormone (rGH) gene was assessed by using a hypomethylating agent, 5-azacytidine, and the iso-schizomeric restriction enzymes MspI and HpaII. 5-Azacytidine increased rGH mRNA 3-8-fold in GH3D6 cells, a subclone of rat pituitary tumor cell lines that expresses one-tenth to one-fifteenth the GH expressed by two other clones, GH3 and GC. The effect was also detected at the level of pre-mRNA. The effect was independent of glucocorticoids and thyroid hormones and was found to be inheritable. The DNA methylation pattern generated by the isoschizomeric restriction enzymes indicated that the HpaII sites in the rGH gene were mostly methylated in GH3D6 cells but mostly unmethylated in GC cells. After treatment with 5-azacytidine, about 22% of these HpaII sites in GH3D6 cells became unmethylated. Thus, DNA methylation correlates inversely with the expression of the rGH gene in these cell lines. However, three other observations indicate that factors in addition to DNA methylation control rGH expression. First, in GC cells, even though most of the HpaII sites are unmethylated, the gene is not fully expressed. Second, in rat hepatoma cells, which do not express GH at all, the GH gene is less methylated than that in GH3D6 cells. Third, within the sensitivities of the assay methods, 5-azacytidine has no effect on the GH gene when it is completely silent. Taken together, the findings indicate that DNA methylation modulates but does not control GH gene expression. It is tempting to speculate that DNA methylation can influence expression only when the gene is committed to express.     

The molecular weight forms of rat growth hormone secreted in response to growth hormone-releasing factor

The different molecular weight forms of immunoreactive growth hormone (irGH) secreted by the anterior pituitary of rats were evaluated during basal and stimulated secretion in vitro and in vivo. Anterior pituitary cells maintained in a monolayer culture system secreted only a 22,000-Da form of GH based on Sephacryl S-200 column chromatography. This was also true for the irGH secreted in response to growth hormone-releasing factor and prostaglandin E2 stimulation. In contrast, the molecular weight forms of irGH found in plasma under basal conditions included an approximately 90,000-Da form as well as the expected 22,000-Da form. The concentrations of both forms increased following growth hormone-releasing factor stimulation although there was a shift in the ratio of the forms secreted. These results suggest that the larger molecular weight forms of rat GH observed in plasma may be the result of some postsecretory process which occurs in blood and suggests a possible regulatory function for the larger molecular weight forms as it pertains to the bioavailability of GH in vivo.     

Growth enhancement of fowls by dietary administration of recombinant yeast cultures containing enriched growth hormone

In present study the methylotrophic yeast, Pichia pastoris, was used to express a recombinant growth hormone (rGH) gene of swine. A synthetic secretion cassette was constructed using the promoter of the alcohol oxidase1 gene (AOX1), and a alpha-factor signal peptide. After electroporatic transformation and zeocin selection, several clones exhibited high levels of rGH protein expression constituting more than 20% of total yeast protein. Over 95% of rGH was shown to be export into the culture supernatant. Yeast transformant containing the highest recombinant growth hormone level (rGH yeast) and native GS115 Pichia pastoris (non-rGH yeast, as a control) were separately cultured, harvested and adsorbed by wheat bran. Yeast cultures of four dosages (0.05, 0.1, 0.2, and 0.4%) were mixed respectively with chick basal diet and fed to simulated country chickens for 9 weeks. The results showed that, when compared to control chicks, the percentage of body weight gain was improved significantly (P<0.05) in chicks fed with diets containing 0.1 or 0.2% rGH-rich yeast culture at brooding stage, and in chicks fed with 0.4% rGH-rich yeast culture at growing stage. The average weight gain in rGH yeast treated groups for the full-term (0 to 63d) and short term (43 to 63d) of growth were 10.6 and 9.4%, respectively, better than the non-rGH yeast control group. These experimental data suggest that the use of rGH-containing yeast as a supplement in fed provided an alternative approach for growth improvement in simulated country chickens.     

Further evaluation of IGF-I responsiveness to ACTH in children affected with IGHD.

In our previous studies we had demonstrated that, in children affected with isolated GH deficiency (IGHD), a short-term recombinant growth hormone (rGH) therapy increases the 11-deoxycortisol (S) secretion and induces an IGF-I responsiveness to the ACTH challenge. The aim of the present study was to further investigate the mechanisms by which IGF-I is secreted after ACTH challenge in children affected with IGHD by correlating IGF-I versus cortisol (F) time courses after ACTH administration. Ten children affected with IGHD were subjected to rGH therapy (4 IU/day subcutaneously) for 10 days. The responsiveness of IGF-I, F and S to the ACTH 1-17 test were evaluated before and at the end of the therapy. No IGF-I response to the ACTH test was recorded in the patients before the rGH treatment, whereas after rGH administration ACTH induced a significant IGF-I release (p < 0.001) which started at the 1st hour, reached a peak value between the 5th and 6th hours and disappeared at the 10th hour. In conclusion, our study confirms that a short-term rGH therapy induces an IGF-I responsiveness to ACTH and helps to better define the kinetics and the mechanism of this IGF-I response to ACTH.     

Effects of varying the position of thyroid hormone response elements within the rat growth hormone promoter: implications for positive and negative regulation by 3,5,3'-triiodothyronine

The thyroid hormone response element (T3RE) of the rat GH (rGH) promoter is located at -188 to -165 relative to the mRNA start site (TSS). Similar sites have been identified in other genes regulated by T3. We have investigated some of these T3REs in positions within the rGH promoter to assess the relative influences of DNA-binding site and position on positive and negative regulation by T3. Synthetic oligonucleotides were used with sequences from the rGH T3RE and proposed negative T3REs (nT3RE) from the rat and human alpha-subunit and rat beta TSH genes. The nT3REs were placed in the background of the wild-type rGH promoter in two positions, at -55 and down-stream of the TSS, with up- and down-mutations of the rGH T3RE. Rat GH T3RE elements were placed 700 basepairs up-stream of a basal rGH promoter and some also at the -55 and TSS positions. Constructions were tested in a transient transfection assay in rat pituitary tumor cells. Two copies of the rGHPAL (palindromic T3RE) placed 700 basepairs up-stream of the rGH promoter conferred 10-fold T3 induction. In the -55 position, the rGHPAL increased T3 induction compared to that in controls, whereas a fragment from the rat and human alpha-subunit gene in the same position reduced induction. Negative T3REs from rat beta TSH and human alpha-subunit reduced T3 induction 50% when placed at the TSS position of a rGH promoter containing an up-mutant T3RE. The T3REPAL placed at the same site increased T3 induction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and secretion of phosphorylated growth hormone by rat pituitary glands in vitro

Rat anterior pituitary glands were incubated in buffered medium, pH 7.4, containing 32Pi. After incubation the tissue and medium were separated and a post-mitochondrial supernate (PMS) of the tissue homogenate was prepared. Gel filtration of the PMS and medium resulted in a radioactive peak which coincided with the elution volume of authentic rat growth hormone (rGH). Polyacrylamide gel electrophoresis of the radioactive peak under denaturing condition resulted in a protein-staining band having the same mobility as authentic rGH. Autoradiography of the gels revealed radioactivity precisely at the position of growth hormone as well as elsewhere. The specific radioactivity of the PMS [32P]GH was estimated to be 5 to 10 times greater than that of tissue [32P]GH. These results indicate that phosphorylated GH is synthesized and secreted by pituitary glands in vitro.     

Growth hormone priming as an adjunct treatment in superovulatory protocols in the ewe alters follicle development but has no effect on ovulation rate

This study investigated the effect of FSH alone and rGH priming followed by FSH treatment on follicle populations, follicular fluid concentrations of components of the IGF system and steroids, and the ovulation rate in sheep. Estrus was synchronized with progestagen sponges. Ewes (n = 10/group) in Group 1 served as untreated controls, while those in Groups 2 to 5 received a standard superovulatory treatment of 1.1 mg i.m. oFSH twice daily for 4 d. In addition, ewes in Groups 3 and 5 were administered rGH (15 mg/d, i.m.) for the 7 d prior to FSH treatment. Groups 1, 2 and 3 were sacrificed just prior to the LH surge; Groups 4 and 5 were allowed to ovulate. Daily plasma samples were collected to monitor GH, IGF-1 and insulin levels. All follicles > or = 1.0 mm from Groups 1, 2 and 3 were counted, and follicular fluid from follicles > or = 2.5 mm was assayed for estradiol, testosterone, IGF-1 and IGFBPs. Compared with the control, treatment with rGH + FSH but not FSH alone increased (P < 0.001) plasma concentrations of GH, IGF-1 and insulin. The mean number of large-(> or = 4.5 mm) and medium-sized (2.5 to 4.0 mm) follicles was increased (P < 0.01), and the mean number of small (< or = 2.0 mm) follicles was decreased (P < 0.001) by FSH treatment. The mean number of medium-sized (2.5 to 4.0 mm) follicles was further increased (P < 0.05) by rGH priming. Estradiol concentration in medium but not in large estrogenic follicles was increased (P < 0.05) by rGH priming, whereas testosterone concentration in estrogenic follicles was not altered. Components of the IGF system in medium-sized estrogenic follicles were similar in all treatment groups; however, in large estrogenic follicles rGH increased IGF-1 concentrations (P < 0.05) and intensity of the 44-42 kDa IGFBP band (P < 0.01). Priming with rGH did not alter superovulatory responses. These results show that rGH priming, when used as an adjunct to FSH treatment in ewes, alters components of the IGF system in large estrogenic follicles and increases the number and physiological maturity of medium-sized follicles in the ovary; it does not however alter ovulation rate responses.