Expression of tuna growth hormone cDNA in Escherichia coli

Summary In order to produce tuna (Thunnus thynnus) growth hormone (GH), expression plasmid (pUES13S) carrying tuna GH cDNA was constructed using a vector (pKK223-3), in which the replication origin was replaced with that of pUC19. The expression of the tuna GH cDNA was greatly affected by the distance between a Shine-Dalgarno (SD) sequence and the initiation codon (ATG) and was most efficient when the distance was adjusted to 13 base pairs (bp). The amount of tuna GH produced by Escherichia coli JM109 with pUES13S was more than 12.5% of the total cytosolic proteins and the product was immunologically identified to be tuna GH (mol. wt. 21 000) by Western blot analysis using tuna GH specific immunoglobulin G (IgG). Another plasmid (pUES13S-2) containing tandemly polymerized tuna GH cDNA was constructed, to improve the productivity of tuna GH. When E. coli JM109 carrying pUES13S-2 was incubated at 40°C, the amount of tuna GH produced reached about 20% of the total cytosolic proteins.     

Molecular cloning and nucleotide sequence of tuna growth hormone cDNA

cDNA for mRNA of tuna growth hormone (GH) was cloned by screening a cDNA library constructed from tuna pituitary gland poly(A)⁺ RNA. The nucleotide sequence of cDNA (911 bases) revealed an open reading frame of 615 nucleotides, including a sequence (51 bases) for a possible secretory protein leader peptide. Noncoding regions were found in the nucleotide sequences up- (5′-terminal: 65 bases) and down- (3′-terminal: 231 bases) stream of the open reading frame. An amino-acid sequence deduced from the nucleotide sequence of the cDNA was identical with that determined in the purified tuna GH. Tuna GH was composed of 187 amino acids, and had a calculated molecular weight of 21275. Amino-acid sequencing showed that there was one possible N-glycosylation site at Asn (Asn-Cys-Thr). Tuna GH showed amino-acid sequence homologies with chum salmon (67%), yellow tail (90%) and with human (32%) growth hormones.     

Purification of carp growth hormone and cloning of the complementary DNA

The growth hormone (GH) was isolated and purified from common carp (Cyprinus carpio) pituitary glands by salt precipitation and HPLC on reverse-phase C18 columns. The carp GH cDNA was synthesized and cloned in Escherichia coli using EcoRI linkers and pBR322 as vector. The positive clones were selected and sequenced. The full-length carp GH cDNA contains 1187 nucleotide basepairs with an open reading frame coding for the precursor form carp GH of 210 amino-acid residues. The partial amino-acid sequence from the protein completely agrees with that derived from the cDNA, with serine as the first residue in mature carp GH preceded by a 22-residue hydrophobic signal peptide. Comparison of the amino-acid sequence of carp GH with those of various species reveals positional identity at 32.4%, 38.8%, 42.0%, 37.2%, 66%, 55% and 49% with GHs of man, rat, duck, bullfrog, salmon, tuna and yellow tail, respectively.     

A Novel Synthesis of l-Pyroglutamyl-glycyl-l-arginine-p-nitroanilide Hydrochloride

Growth hormone (GH) was purified from pituitary glands of tuna (Thunnus albacares). The yield of this hormone was 4mg/g wet tissue. The hormone had a molecular weight of 21,000 and an isoelectric point of 7.1. The partial amino acid sequence including N-terminal Gin, which was modified to pyroglutamate, was established by analyzing peptide fragments generated by chemical and enzymatic treatments. Intraperitoneal injection of tuna GH at doses of 0.1 and 1 μ body weight at 7 day intervals resulted in a significant increase in body weight and length of juvenile rainbow trout (Salmo gairdneri). The GH-treated fish had a 3-fold higher growth rate and a 1.6-fold higher food conversion efficiency than the control fish.     

Phosphorylation of highly purified growth hormone receptors by a growth hormone receptor-associated tyrosine kinase

We have shown previously that growth hormone (GH) promotes the phosphorylation of its receptor on tyrosyl residues (Foster, C. M., Shafer, J. A., Rozsa, F. W., Wang, X., Lewis, S. D., Renken, D. A., Natale, J. E., Schwartz, J., and Carter-Su, C. (1988) Biochemistry 27, 326-334). In the present study, we investigated the possibility that a tyrosine kinase is specifically associated with the GH receptor. GH-receptor complexes were first partially purified from GH-treated 3T3-F442A fibroblasts, a GH-responsive cell, by immunoprecipitation using anti-GH antiserum. 35S-Labeled proteins of Mr = 105,000-125,000 were observed in the immunoprecipitate from GH-treated cells labeled metabolically with 35S-amino-acids. These proteins were not observed in immunoprecipitates from cells not exposed to GH or when non-immune serum replaced the anti-GH antiserum, consistent with the proteins being GH receptors. GH receptors appeared to be phosphorylated, as evidenced by the presence of 32P-labeled bands, comigrating with the 105-125 kDa 35S-labeled proteins, in the immunoprecipitate of GH-treated cells labeled metabolically with [32P]Pi. When partially purified GH receptor preparation was incubated with [gamma-32P]ATP (7-15 microM) for 10 min at 30 degrees C in the presence of MnCl2, a protein of Mr = 121,000 was phosphorylated exclusively on tyrosyl residues. As expected for the GH receptor, this protein was not observed in immunoprecipitates when cells had not been treated with GH nor when non-immune serum replaced the anti-GH antiserum. GH-receptor complexes were also purified to near homogeneity by sequential immunoprecipitation with phosphotyrosyl-binding antibody followed by anti-GH antiserum. When cells were labeled metabolically with 35S-amino acids, the 35S label migrated almost exclusively as an Mr = 105,000-125,000 protein. This protein also incorporated 32P into tyrosyl residues when incubated in solution with [gamma-32P]ATP. These results show that highly purified GH receptor preparations undergo tyrosyl phosphorylation, suggesting that either the GH receptor itself is a tyrosine kinase or is tightly associated with a tyrosine kinase.     

Purification and partial characterization of a nonprimate growth hormone receptor.

Pregnant rabbit liver membranes have been shown to possess two types of receptors by displacement analysis, a growth hormone (GH) receptor which binds bovine growth hormone with an affinity constant (KA) of 3 x 10(9) M-1 and ovine prolactin with a KA of 3 x 10(8) M-1, and a prolactin (Prl)-specific receptor which binds ovine prolactin with a KA of 5 x 10(9) M-1. The prolactin-specific receptor when solubilized with Triton exhibits a 4-fold increase in the its KA while the KA of the growth hormone receptor decreases slightly to 2 x 10(9) M-1 after solubilization. The 10-fold difference in affinity which results has been exploited to facilitate the separation of these two receptors by differential affinity chromatography on human growth hormone (hGH) affinity gels. The growth hormone receptor is eluted from the gel with 4 M urea while 5 M MgCl2 is required to elute the prolactin receptor. Conditions of affinity chromatography have been optimized, and further purification of the GH receptor by preparative isoelectric focusing and Sepharose 6B gel filtration resulted in a more than 8000-fold purification of the receptor. This material had a Stokes radius of 62 A, consistent with a molecular weight of 300,000 and gave one main band (75,000 to 80,000) and two minor bands on sodium dodecyl sulfate (SDS) polyacrylamide gels, which could be interpreted as indicating a tetrameric receptor. The GH receptor was shown to be a sialoglycoprotein (or closely associated with sialoglycoprotein) by analytical isoelectric focusing with an isoelectric point of 4.6. Specificity studies with the highly purified receptor confirmed the initial hypothesis that this receptor is capable of binding bovine growth hormone (bGH) with high affinity and ovine prolactin (oPrl) with low affinity, in contrast to the prolactin-specific receptor.     

Periplasmic production of correctly processed human growth hormone in Escherichia coli: natural and bacterial signal sequences are interchangeable

We have studied the synthesis, secretion, and processing of human growth hormone (hGH) in Escherichia coli transformed with plasmids engineered for the expression of hGH as a secreted product. In one plasmid, pPreHGH207-2, the coding sequence of the natural hGH precursor (pre-hGH) is placed under the control of the E. coli trp promoter. In a second plasmid, pAPH-1, a DNA fragment containing the E. coli alkaline phosphatase promoter and signal sequence codons is fused to the mature hGH coding sequence (pho-hGH). Most of the hGH was present in the osmotic shock fluids of E. coli cells containing either plasmid, indicating transport to the periplasmic space. Amino acid sequencing of the N termini of the pre-hGH and pho-hGH gene products revealed that both were processed correctly. Electrophoretic analysis of these polypeptides on reducing and nonreducing sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels indicates that periplasmic hGH is monomeric and contains the same two disulfide bonds as authentic hGH.     

Purification of duck growth hormone and cloning of the complementary DNA

Duck growth hormone (GH) was isolated and purified from duck pituitaries by salt precipitation and HPLC on reverse-phase C18 columns. The duck GH was homogeneous as shown by SDS-polyacrylamide gel electrophoresis with a molecular weight of 22,000. The cDNA was synthesized and cloned in Escherichia coli using EcoRI linkers and pBR322 as vector. The positive clones were selected and sequenced. The full-length duck GH cDNA contains 820 nucleotide pairs with an open reading frame coding for the precursor form duck GH of 216 amino-acid residues. The partial amino-acid sequence from the protein completely agrees with that derived from the cDNA, with Phe as the first residue in mature duck GH preceded by a 27-residue hydrophobic signal peptide. The duck GH is almost completely homologous to the chicken GH, with only three conservative substitutions (Ser for Thr, His for Tyr and Lys for Arg) and one deletion (Ala) in the duck GH sequence. Comparison of amino-acid sequence of duck GH with that of various species reveals 56%, 73% and 40% homologies with GHs of human, rat and salmon, respectively.     

Effect of mammalian growth hormone and prolactin on the growth of hypophysectomized chickens

Body weight gain and shank-toe growth during a 26-day treatment period following hypophysectomy were 55 and 46%, respectively, of control values, but the body weight gain was unaffected and bone growth only slightly reduced when the hypophysectomized chickens were fed a low dose of corticosterone (5 ppm). Bovine growth hormone (0.5 mg GH/kg body wt/day for 18 days) enhanced body weight gain and shank-toe length increase (an estimate of bone growth) by 46 and 33%, respectively, compared to the growth of hypophysectomized chickens receiving only corticosterone. These same endpoints were increased approximately 24% after ovine growth hormone treatment in hypophysectomized chickens not receiving corticosterone. Body weight gain during 18 days of treatment with bovine prolactin (0.5 mg PRL/kg/day) was 27% greater than the value for corticosterone-treated hypophysectomized chickens, but bone growth was unaffected. The mammalian GH preparations increased heart weight of the hypophysectomized chickens (25-29%), but pectoralis muscle weight was unaffected. GH treatment enhanced thymal weights by 71% in corticosterone-treated hypophysectomized chickens, and by 93% in hypophysectomized animals not receiving corticosterone. GH had no significant effect on bursal weights, and PRL had no effect on either of these lymphoid organ weights in corticosterone-treated hypophysectomized chickens. GH increased liver and adipose tissue weights considerably more than the large increases that followed treatment of hypophysectomized chickens with corticosterone alone (69 and 126% greater, respectively), but had no effect on these endpoints in hypophysectomized chickens not receiving corticosterone. PRL also greatly increased liver and adipose tissue weights in corticosterone-treated hypophysectomized chickens (79 and 75%, respectively). These results provide evidence that mammalian GH enhances body weight gain, bone growth, and the growth of several organs in the hypophysectomized chicken. Mammalian PRL increased body weight gain, liver weight, and adipose tissue weight in corticosterone-treated hypophysectomized chickens, but did not influence bone growth or the weights of the heart, pectoralis, thymi, or bursa.     

The macrophage-activating properties of growth hormone

1. We compared the ability of growth hormone (GH) and a well-characterized macrophage-activating factor, interferon-gamma (IFN-gamma) to activate highly purified populations of alveolar macrophages. Both GH and IFN-gamma primed macrophages triggered with opsonized zymosan to secrete superoxide anion (O2-) in vitro, but IFN-gamma was effective at a 40-fold lower concentration. Antibody blocking studies demonstrated that the priming activity of GH was independent of IFN-gamma, and the activity of IFN-gamma was distinct from that of GH. 2. Both IFN-gamma and GH increased the capability of macrophages to kill Pasteurella multocida in vitro. 3. Hypophysectomized rats challenged with Salmonella typhimurium were significantly protected by injections of either GH or recombinant rat IFN-gamma in vivo compared to vehicle-treated controls, and the protective effect of GH was increased by incorporation into liposomes. 4. Insulin-like growth factor-I (IGF-I) also primed alveolar macrophages in vitro, which is consistent with the idea that the protective effects of GH in vivo might be mediated by augmenting the synthesis of IGF-I. These data support the concept of reciprocal systems of communication between the neuroendocrine and immune systems.     

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.     

Site-directed mutations of human growth hormone that selectively modify its lactogenic activity and binding properties

Two novel analogs of human (h) GH, 1) Des-7-hGH (Arg8Met, Asp11Ala) in which the Arg8 was substituted by Met and Asp11 by Ala, and 2) bovine (b) GH/hGH hybrid II (MetAla 1-13/14-191, Ala11Asp) composed of 13 N-terminal amino acid of bGH and elongated by two amino acids (Met-Ala-1-13) and 14-191 amino acids of hGH, were constructed and expressed in Escherichia coli. CD spectra indicated that the alpha-helix content of the purified proteins was similar to that of the native hormone. Both analogs retained their full ability to stimulate the proliferation of Nb2 lymphoma cells, and their binding to the lactogen receptors in homogenate of Nb2 cells and in microsomal fraction from bovine lactating mammary gland was only slightly reduced. However, their ability to bind to the somatogen receptors in intact IM-9 lymphocytes and bovine liver was reduced by 7- to 11-fold (bGH/hGH hybrid II) and 20- to 30-fold (Des-7-hGH). Both analogs were able to down-regulate the respective lactogen and somatogen receptors in intact Nb2 and IM-9 cells. The galactopoietic activity of both analogs in the lactating bovine mammary explants bioassay was almost completely abolished, and the bGH/hGH hybrid II exhibited a remarkable antagonistic activity. These results further indicate that the lactogen receptors in different species or organs are not identical. We have shown that the new recombinant analogs of hGH that recognize both somatogen and lactogen receptors but have modified postreceptor effects are helpful in elucidating these differences.     

Induction of growth hormone (GH) mRNA by pulsatile GH-releasing hormone in rats is pattern specific

Growth hormone-releasing hormone (GHRH) is a main inducer of growth hormone (GH) pulses in most species studied to date. There is no information regarding the pattern of GHRH secretion as a regulator of GH gene expression. We investigated the roles of the parameters of exogenous GHRH administration (frequency, amplitude, and total amount) upon induction of pituitary GH mRNA, GH content, and somatic growth in the female rat. Continuous GHRH infusions were ineffective in altering GH mRNA levels, GH stores, or weight gain. Changing GHRH pulse amplitude between 4, 8, and 16 microg/kg at a constant frequency (Q3.0 h) was only moderately effective in augmenting GH mRNA levels, whereas the 8 microg/kg and 16 microg/kg dosages stimulated weight gain by as much as 60%. When given at a 1.5-h frequency, GHRH doubled the amount of GH mRNA, elevated pituitary GH stores, and stimulated body weight gain. In the rat model, pulsatile but not continuous GHRH administration is effective in inducing pituitary GH mRNA and GH content as well as somatic growth. These studies suggest that the greater growth rate, pituitary mRNA levels, and GH stores seen in male compared with female rats are likely mediated, in part, by the endogenous episodic GHRH secretory pattern present in males.     

The effect of cyclosporine administration on growth hormone release and serum concentrations of insulin-like growth factor-I in male rats.

The aim of this work was to study the effect of cyclosporine on the somatotropic axis. Accordingly, growth hormone (GH) secretion, circulating insulin-like growth factor I (IGF-I) and IGF binding proteins (IGFBPs) in response to cyclosporin A (CsA) treatment were examined in adult male Wistar rats. Cyclosporine administration (5, 10 or 20 mg/Kg daily) over 8 days did not modify the body weight, but it did decrease serum concentration of corticosterone and increased serum IGF-I and GH levels. Rats treated with 5 and 10 mg/Kg of cyclosporine had similar levels of serum IGFBPs to control rats, but there was an increase in circulating IGFBP-3 and IGFPB-1,2 in the group treated with 20 mg/Kg of CsA. The increase in circulating GH correlates with a decrease in pituitary GH content in CsA treated rats, with no modification in hypothalamic somatostatin content, suggesting an increase in pituitary GH release. In order to test this hypothesis, anterior pituitary cell cultures were exposed to different CsA concentrations during a 4 h incubation period. Cyclosporine increased GH secretion in cultured pituitary cells (p<0.05). These data suggest that cyclosporine increases circulating IGF-I and GH by stimulating pituitary GH release.     

Differences in pathological findings and growth hormone responses in patients with growth hormone-producing pituitary adenoma.

Plasma growth hormone (GH) responses to various stimuli were examined in 21 patients with GH-producing pituitary adenomas, classified into three types by the immunohistochemistry of cytokeratin and the glycoprotein hormone alpha-subunit distribution. Seven type 1 adenomas were exclusively composed of cells in which the cytokeratin formed a dot-like pattern; they were chromophobic to hematoxylin and eosin (H&E), occasionally positive for GH, and almost completely negative for the alpha-subunit. Thirteen type 2 adenomas were composed of cells with cytokeratin that had a perinuclear distribution; they were eosinophilic to H&E, and diffusely positive for both GH and the alpha-subunit. One patient had a type 3 adenoma which had a mixed pattern of intracellular cytokeratin distribution and was chromophobic and eosinophilic to H&E. Clinically, type 1 is characterized by earlier onset, larger tumor size, and more frequent aggressive extension. Paradoxical GH responses to TRH and OGTT were seen in 1 of 6 patients (16.7%) of type 1 and 8 of 9 patients (88.9%) of type 2, and 0% of type 1 and 62.5% of type 2, respectively. Type 2 cases showed higher plasma GH response to GH-releasing hormone, and a tendency to greater suppression of plasma GH by bromocriptine compared with type 1. Octreotide acetate administration revealed that the nadir/basal ratio of plasma GH levels was 42.9 +/- 6.6% in type 1 and 13.5 +/- 5.8% in type 2. These results suggest that there is a pathophysiological difference between these two distinct types of GH-producing pituitary adenomas.     

Cloning and nucleotide sequencing of sheep growth hormone cDNA.

cDNA was prepared from the mRNA isolated from sheep anterior pituitary glands. On cloning cDNA in E. coli, a clone coding full sequence of sheep pre-growth hormone was determined. The sequence for the sheep growth hormone (GH) is in agreement with the amino acid sequence of the protein determined previously except for the asparagine residue at position 99 rather than aspartic acid and the arginine residue at position 146 in place of threonine. The cDNA sequence presented is also in accordance with the genomic sequence for the sheep GH gene that has been reported.     

Phosphorylation of chicken growth hormone

The possibility that chicken growth hormone (cGH) can be phosphorylated has been examined. Both native and biosynthetic cGH were phosphorylated by cAMP-dependent protein kinase (and gamma -32P-ATP). The extent of phosphorylation was however less than that observed with ovine prolactin. Under the conditions employed, glycosylated cGH was not phosphorylated. Chicken anterior pituitary cells in primary culture were incubated in the presence of 32P-phosphate. Radioactive phosphate was incorporated in vitro into the fraction immunoprecipitable with antisera against cGH. Incorporation was increased with cell number and time of incubation. The presence of GH releasing factor (GRF) increased the release of 32P-phosphate labelled immunoprecipitable GH into the incubation media but not content of immunoprecipitable GH in the cells. The molecular weight of the phosphorylated immunoreactive cGH in the cells corresponded to cGH dimer.     

Biological characterization of purified native 20-kDa human growth hormone

Because of the propensity of the 20-kDa variant of human growth hormone (GH) to aggregate with itself and with 22-kDa human GH, it has been difficult to prepare monomeric 20-kDa GH in highly purified form. This has been a major complicating factor in determining whether 20-kDa GH has a biological activity profile distinct from that of 22-kDa GH. In the present study, native 20-kDa GH was isolated from a human GH dimer concentrate and purified by a procedure that included column electrophoresis in agarose suspension as a final separation step. This procedure yielded highly purified monomeric 20-kDa GH, which was contaminated to an extent of less than 1% with 22-kDa GH, and which exhibited only a small degree of dimerization upon storage. The native 20-kDa GH was quite active in stimulating growth in hypophysectomized rats, when growth was assessed by body weight gain, longitudinal bone growth, the stimulation of sulfation of cartilage, and the elevation of serum IGF-1 level. However, in all of these growth assays, the 20-kDa GH was somewhat less active than the native 22-kDa GH to which it was compared; e.g., in the body weight gain and longitudinal bone growth assays, it had an estimated potency of 0.6 relative to the 22-kDa GH. The 20-kDa GH exhibited substantial diabetogenic activity when tested for the ability to raise fasting blood glucose concentration and to impair glucose tolerance in ob/ob mice. Also, the native 20-kDa GH had significant in vitro insulin-like activity, although its potency was approximately 20% that of the native 22-kDa GH to which it was compared. Thus, the biological activity profile of native 20-kDa GH differs from that of 22-kDa GH primarily in that insulin-like activity is markedly attenuated.