A Moloney MLV-rat somatotropin fusion gene produces biologically active somatotropin in a transgenic pig

Expression of a Moloney murine leukemia virus (MLV) rat somatotropin fusion gene was examined in a transgenic pig. The fusion gene was integrated in a single site within the genome in a tandem array with approximately eight copies per cell. The integrated in a single site within the genome in a tandem array with approximately eight copies per cell. The integrated MLV-rat somatotropin fusion gene produced high levels of circulating rat somatotropin and resulted in an elevation in the circulating levels of insulin-like growth factor I. Although there was no increase in the rate of growth of the transgenic animal during the rapid growth phase, several phenotypic changes were evident. Skeletal growth was markedly increased and fat deposition was reduced throughout the animal. Blood glucose levels were elevated without ketosis. Northern blot analyses of rat somatotropin RNA revealed that expression of the fusion gene was highest in the spleen, lung, intestine, lymph nodes, and bone marrow. These results show that the MLV promoter can be used to express high levels of biologically active rat somatotropin in transgenic swine.     

Conformation of equine pituitary somatotropin

Equine pituitary somatotropin (growth hormone) has been studied by zero-order and second-order absorption spectroscopy, and by circular dichroism. Difference absorption spectra have also been generated during proteolytic digestion of the hormone. The molar extinction coefficient of the native protein was found to be 16,050 +/- 330 M-1 cm-1 at 278.1 nm. Comparison of the conformations of equine somatotropin and somatotropins isolated from several other mammalian species indicates a close structural relationship between these molecules. With the increasing number of species which have been studied, it is becoming evident that with regard to conformation, the somatotropins can be subdivided into at least three major groups.     

Conformation of sturgeon pituitary somatotropin

Pituitary somatotropin (growth hormone) from the sturgeon (Acipenser gulden-stadti) has been studied by zero-order and second-order absorption spectroscopy, as well as by circular dichroism. Difference absorption spectra have also been generated during proteolytic digestion of the hormone. The molar extinction coefficient of the native protein was found to be 15,000 +/- 110 M-1 cm-1 at 278.5 nm. Comparison of the conformations of sturgeon somatotropin and somatotropins isolated from several mammalian species, including bovine and human, indicates a close relationship between these molecules. Such similarities may be related to the relatively high biopotency of this fish hormone in mammalian assay systems.     

Multiple forms of pig somatotropin]

The multiple forms of pig somatotropin were investigated. Four monomer hormone variants were isolated and some of their physico-chemical characteristics were determined. It is shown that they differ in their molecular mass, N-terminal amino acid residues and amino acid composition. The results of work permit supposing that the identified somatotropin forms are controlled by two different genes. The age dependence of spectrum of somatotropin monomer forms is demonstrated.     

The conformation of monkey pituitary somatotropin

Monkey pituitary somatotropin has been studied by zero-order, second-order, and circular dichroism spectroscopy. Difference absorption spectra have also been generated during proteolytic digestion of the hormone. The molar extinction coefficient of the native protein was found to be 23,800 +/- 550 (M-1 cm-1) at 276.6 nm. A comparison of the conformations of monkey and human pituitary somatotropins indicates a close relationship between the two molecules, including alpha-helix contents of 55 +/- 5%.     

Polymorphism in porcine somatotropin cDNA sequences

Three new full-length cDNAs coding for porcine somatotropin (PST) have been cloned. The sequence data indicate a high degree of polymorphism in the PST sequence. All six known PST sequences are different.     

Polymorphism in porcine somatotropin cDNA sequences

Summary. Three new full-length cDNAs coding for porcine somatotropin (PST) have been cloned. The sequence data indicate a high degree of polymorphism in the PST sequence. All six known PST sequences are different.     

Purification and characterization of pituitary bovine somatotropin

Bovine somatotropin (bST) has been isolated from pituitary glands and compared in a variety of chemical analyses and bioassays with somatotropin derived from recombinant Escherichia coli. Comparison of pituitary extracts and purified bST by Western blot analysis of two-dimensional gels suggested that the immunoreactive somatotropin species present in the extract were also present in the purified material, with no significant losses or degradation as a result of the purification method. NH2-terminal sequence analysis indicated the presence of equal quantities of Ala-Phe-Pro-Ala-Met-Ser-Leu-Ser- and Phe-Pro-Ala-Met-Ser-Leu-Ser- sequences. The Met-Ser-Leu-Ser-NH2-terminal sequence, a degradation product observed in NIH standard lots, was not detected. Assay of bioactivity in a bovine liver receptor-binding assay and in a female rat growth assay showed pituitary bST and recombinant methionyl-bovine somatotropin to be equipotent. Tryptic maps and sequence analysis of pituitary-derived somatotropin suggest the presence of isoaspartate derivatization at Asp128.     

High-performance tryptic mapping of recombinant bovine somatotropin

Experiments are described that have lead to the development of a highly reproducible tryptic map of recombinant DNA derived bovine somatotropin (rbSt). Tryptic digestion of rbSt at 37 degrees C results in the formation of a precipitate. Preliminary characterization of the precipitate suggests that its formation is due to the association of intermediate tryptic fragments. An examination of the temperature dependence of the digestion has revealed that precipitate formation is inhibited when digestion is performed at 10 degrees C or less. The combination of a 5-mg sample, the use of highly purified trypsin, and digestion at 5 degrees C generate a tryptic map that exhibits an average 1.3% RSD (0.5-3.6%) for all anticipated fragments. Validation studies demonstrate that while the peak response precision is rugged to daily variation of operators or chromatographic systems, the fragment retention is not. This dictates that peaks be assigned by qualitative pattern recognition. Assay ruggedness in the peak response domain allows for the implementation of quantitative methods for the comparison of rbSt reference standard and sample tryptic maps. The assay is linear for all anticipated fragments within 50-150% of the operating range. Specificity is established by assay of pituitary somatotropins from other species and rbSt analogs produced by site-specific mutagenesis. The data demonstrate that all single amino acid substitutions examined are identified by using the technique. Assay sensitivity is validated for selected tryptic fragments through analysis of reference standard digests spiked with known amounts of rbSt analog digests. The data indicate that potential impurities of 3.2, 2.0, and 4.5% can be quantitated with statistical confidence in the tryptic fragments T1, T10, and T23 + 25, respectively.     

Human somatotropin. Reaction with hydrogen peroxide

Two methionine-modified derivatives of human somatotropin have been prepared by oxidation of the methionines with H₂O₂ to sulfoxide. The monomeric derivatives were characterized by exclusion chromatography, amino acid composition, circular dichroism spectra, relative rates of tryptic digestion, and biological property. Partially oxidized somatotropin, with two of its three methionines oxidized, was found to be similar to the native hormone by all criteria examined except that the growth promoting potency was reduced to 25% of the native hormone. The unoxidized methionine in this derivative was found to be the residue at position 170. The derivative in which all of the methionines had been oxidized showed major changes in all physical parameters examined as well as significant loss of the growth-promoting activity.     

Comparative investigations of human and porcine somatotropin

Human and porcine somatotropin were compared by different methods. Both hormones show biological activity in the Greenspan assay. Molecular masses, N- and C-terminal amino acids as well as the chromatographic patterns on ion exchange cellulose at room temperature are identical. Chromatography in the cold results in different behaviour of the two hormones. Polyacrylamide gel electrophoresis and electrofocusing also show characteristic differences. The quantitative amino acid analyses of the two hormones are different. The N-terminal amino acid sequences estimated up to position 20 show identity only in 13 positions.     

Human somatotropin 61. A simple method to predict potential of somatotropin fragments for noncovalent recombination by radioreceptor assay

Different mixtures of reduced-alkylated thrombin fragments of human and sheep somatotropin have been tested for binding affinity to liver membranes. The bioassay data correlated well with the abilities of the fragments to form noncovalent recombinants as shown in separate biochemical studies.     

A novel concatenated dimer of recombinant bovine somatotropin

A novel protein concatenated dimer structure was generated during the folding/oxidation of inclusion bodies of recombinant bovine somatotropin synthesized inEscherichia coli. The structure of this dimeric molecule was determined by peptide mapping with trypsin, and limited proteolysis by thrombin. Peptide mapping demonstrated that the two disulfide pairs in bovine somatotropin dimer were identical to those in monomer. Limited proteolysis with thrombin resulted in the cleavage of only a single peptide bond between arginine-132 and alanine-133 in bovine somatotropin dimer. This single peptide bond cleavage was sufficient to convert this dimer to a monomeric molecular weight species as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and HPLC. Since the single cleaved peptide bond is present in the large disulfide loop of bovine somatotropin, these data demonstrate that the dimeric molecule exists as a novel concatenated structure through the interlocking of the disulfide loops of this protein.