Effects of pre- and pro-sequence of thaumatin on the secretion by Pichia pastoris

Thaumatin is a 22-kDa sweet-tasting protein containing eight disulfide bonds. When thaumatin is expressed in Pichia pastoris using the thaumatin cDNA fused with both the alpha-factor signal sequence and the Kex2 protease cleavage site from Saccharomyces cerevisiae, the N-terminal sequence of the secreted thaumatin molecule is not processed correctly. To examine the role of the thaumatin cDNA-encoded N-terminal pre-sequence and C-terminal pro-sequence on the processing of thaumatin and efficiency of thaumatin production in P. pastoris, four expression plasmids with different pre-sequence and pro-sequence were constructed and transformed into P. pastoris. The transformants containing pre-thaumatin gene that has the native plant signal, secreted thaumatin molecules in the medium. The N-terminal amino acid sequence of the secreted thaumatin molecule was processed correctly. The production yield of thaumatin was not affected by the C-terminal pro-sequence, and the pro-sequence was not processed in P. pastoris, indicating that pro-sequence is not necessary for thaumatin synthesis.     

Germination-specific cortex-lytic enzymes from Clostridium perfringens S40 spores: time of synthesis, precursor structure and regulation of enzymatic activity

Germination-specific enzymes, an amidase and a muramidase, of Clostridium perfringens S40 were synthesized at the time of forespore formation during sporulation. The amidase had a unique precursor structure consisting of four domains: the N-terminal pre-sequence, the N-terminal pro-sequence, mature enzyme and the C-terminal pro-sequence. The N-terminal pre-sequence and the C-terminal pro-sequence were sequentially processed at the time of development of phase-bright spores, and the resulting inactive pro-enzyme was activated by cleavage of the N-terminal pro-sequence with a specific protease during germination. A possible mechanism for the regulation of activity of muramidase, which is produced as a mature form and does not need processing for activation, is presented.     

Secretory expression of the human serum albumin gene in the yeast, Saccharomyces cerevisiae

We have fused a cDNA gene encoding mature human serum albumin (HSA) to several secretory leader-encoding sequences. The hybrid genes were cloned into an episomal vector under the control of several yeast promoters and then introduced into yeast cells. The GAL1 promoter in combination with either the native HSA pre-sequence or a modified HSA pre-sequence gave the highest production of immunoreactive HSA, 90 mg/liter being reached in a shake flask culture. The invertase pre-sequence, the mating factor alpha 1 prepro-sequence, and the modified HSA pre-sequence directed accurate processing. In contrast, the chicken lysozyme pre-sequence and the native HSA pre-sequence directed incorrect processing. Episomal vectors were unstable within the host cells under non-selective culture conditions. To improve the plasmid stability, the hybrid genes were incorporated into an integrative vector. Transformants carrying multicopies of the plasmid integrated at the LEU2 locus stably secreted HSA. The highest yield of 65 mg/liter in a shake flask culture was obtained with the combination of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter and the modified HSA pre-sequence. By constructing transformed strains containing multicopies of plasmids integrated at both the chromosome LEU2 and HIS4 loci, we have obtained a stable strain that continuously secretes as much as 85 mg HSA per liter of culture medium.     

Requirement of pro-sequence for the production of active subtilisin E in Escherichia coli

Subtilisin E, an alkaline serine protease of Bacillus subtilis 168, is first produced as a precursor, pre-pro-subtilisin, which consists of a signal peptide for protein secretion (pre-sequence) and a peptide extension of 77 amino acid residues (pro-sequence) between the signal peptide and mature subtilisin. When the entire coding region for pre-pro-subtilisin E was cloned into an Escherichia coli expression vector, active mature subtilisin E was secreted into the periplasmic space. When the pre-sequence was replaced with the E. coli OmpA signal peptide, active subtilisin E was also produced. When the OmpA signal peptide was directly fused to the mature subtilisin sequence, no protease activity was detected, although this product had the identical primary structure as subtilisin E as a result of cleavage of the OmpA signal peptide and was produced at a level of approximately 10% of total cellular protein. When the OmpA signal peptide was fused to the 15th or 44th amino acid residue from the amino terminus of the pro-sequence, active subtilisin was also not produced. These results indicate that the pro-sequence of pre-pro-subtilisin plays an important role in the formation of enzymatically active subtilisin. It is proposed that the pro-sequence is essential for guiding appropriate folding of the enzymatically active conformation of subtilisin E.     

人甲状旁腺激素在大肠杆菌中的表达及鉴定

化学合成人甲状旁腺激素（hPTH）全长基因,克隆到大肠杆菌表达载体pBV220和pET22b中,获得了高表达。经破菌、阳离子交换层析、反相层析纯化获得了纯度大于95%的纯品。N端测序、质谱分析结果表明重组hPTH 结果完整,N端无Met或fMet。生物活性试验证明重组hPTH具有激活腺苷酸环化酶、增加骨质量和骨密度等作用。     

Isolation and Characterization of a cDNA That Encodes a Novel Proteinase Inhibitor I from a Tobacco Genetic Tumor

We have isolated a cDNA clone, designated GTI, by screeninga tobacco genetic tumor cDNA library with a tumor-specific "subtracted"cDNA probe. The cDNA contained the entire coding sequence fora 94-amino-acid polypeptide that exhibited significant homologyto members of the proteinase inhibitor I family from tomatoand potato. The predicted protein has a pre-sequence of 22 aminoacids but lacks a pro-sequence, unlike genes for proteinaseinhibitor I isolated to date. Furthermore, the protein encodedby GTI cDNA has a novel reactive site, having glutamine as theP₁ reactive residue. These results suggest that the GTI proteinis a novel member of the proteinase inhibitor I family. ThemRNA for GTI accumulated at a high level but only transientlyafter the wounding of tobacco plants. Thus, it appears thatthe GTI protein has a function that is related to the protectionof tissues against damage due to wounding. (Received July 22, 1992; Accepted November 3, 1992)     

重组人甲状旁腺激素肽（1-34）在大肠杆菌中的高效融合表达及纯化

人工合成人甲状旁腺激素1-34肽段 (PTH1-34)的cDNA序列,克隆到大肠杆菌蛋白表达载体pThioHis中,获得了高表达菌株。经发酵、破菌、金属鳌合层析、反相层析和凝胶层析后获得了纯度大于95%的hPTH1-34。hPTH1-34肽N端测序和质谱分子量测定结果与天然PTH1-34一致。生物学活性研究表明,hPTH1-34在体外具有刺激腺苷酸环化酶的作用。     

Increased biological activity of a recombinant factor IX variant carrying alanine at position +1

In attempts to improve the post-translational modification and processing of recombinant factor IX (FIX) we have altered the cDNA sequence encoding pre-pro-FIX using site-directed mutagenesis and have expressed the variant cDNAs in BHK21 cells using a vaccinia-virus-derived vector. We find that substitution of the tyrosine residue at +1 for an alanine increases the biological activity of the recombinant molecules 2-fold. On the other hand, substitution of the proline at -3 for a valine results in no significant change to the specific activity of the protein. Other alterations to the N-terminus of the FIX proteins, in attempts to mimic other vitamin-K-dependent proteins, result in the failure to produce a secreted polypeptide. N-terminal sequence analysis of purified recombinant molecules reveals a correlation between specific activity and the efficiency of correct pro-sequence cleavage. gamma-Carboxylation analysis of purified recombinant proteins indicates that each molecule including unmutated FIX is completely gamma-carboxylated in this system. Thus the observed increase in biological activity of FIX variants containing an alanine at position +1 is not due to increased gamma-carboxylation but, at least in part, to more efficient pro-peptide cleavage.     

Efficient secretion of human parathyroid hormone by Saccharomyces cerevisiae

A cDNA encoding mature human parathyroid hormone (hPTH) was expressed in Saccharomyces cerevisiae, after fusion to the prepro region of yeast mating factor alpha (MF alpha). Radioimmunoassay showed high levels of hPTH immunoreactive material in the growth medium (up to 10 micrograms/ml). More than 95% of the immunoreactive material was found extracellularly as multiple forms of hormone peptides. Three internal cleavage sites were identified in the hPTH molecule. The major cleavage site, after a pair of basic amino acids (aa) (Arg25Lys26 decreases Lys27), resembles that recognized by the KEX2 gene product on which the MF alpha expression-secretion system depends. The use of a protease-deficient yeast strain and the addition of high concentrations of aa to the growth medium, however, not only changed the peptide pattern, but also resulted in a significant increase in the yield of intact hPTH (1-84) (more than 20% of the total amount of immunoreactive material). The secreted hPTH (1-84) migrates like a hPTH standard in two different gel-electrophoretic systems, co-elutes with standard hPTH on reverse-phase high-performance liquid chromatography, reacts with two hPTH antibodies raised against different parts of the peptide, has a correct N-terminal aa sequence, and has full biological activity in a hormone-sensitive osteoblast adenylate cyclase assay.     

Effects of<Emphasis Type="Italic">Nigella sativa</Emphasis> and human parathyroid hormone on bone mass and strength in diabetic rats

Osteoporosis is a major complication in patients with diabetes mellitus (DM), particularly in those with insulin dependency. Recently, many therapeutic effects ofNigella sativa L. (NS) extracts have been exhibited such as anti-inflammatory, antitumor, and antidiabetic with clinical and experimental studies. Mechanical strength in the femur and vertebrae increases with human parathyroid hormone (hPTH) treatment. The aim of the present study was to test the hypothesis that combined treatment with NS and hPTH is more effective than treatment with NS or hPTH alone in improving bone mass, connectivity, and biomechanical behavior using the finite element method (FEM) in insulin-dependent diabetic rats. In the mechanical analysis, five rat bones (control, diabetic diabetic NS treated, diabetic hPTH treated, and diabetic NS + hPTH treated) have been studied for bending analysis using the finite element analysis program ANSYS. Combined treatment of NS and hPTH was more effective on bone histomorphometry and mechanical strength than treatment with NS or hPTH alone for streptozotocin-induced diabetic osteopenia, which notably decreased bone volume.     

Disuse in adult male rats attenuates the bone anabolic response to a therapeutic dose of parathyroid hormone.

Intermittent treatment with parathyroid hormone (PTH) increases bone formation and prevents bone loss in hindlimb-unloaded (HLU) rats. However, the mechanisms of action of PTH are incompletely known. To explore possible interactions between weight bearing and PTH, we treated 6-mo-old weight-bearing and HLU rats with a human therapeutic dose (1 microg.kg(-1).day(-1)) of human PTH(1-34) (hPTH). Cortical and cancellous bone formation was measured in tibia at the diaphysis proximal to the tibia-fibula synostosis and at the proximal metaphysis, respectively. Two weeks of hindlimb unloading resulted in a dramatic decrease in the rate of bone formation at both skeletal sites, which was prevented by PTH treatment at the cancellous site only. In contrast, PTH treatment increased cortical as well as cancellous bone formation in weight-bearing rats. Two-way ANOVA revealed that hPTH and HLU had independent and opposite effects on all histomorphometric indexes of bone formation [mineral apposition rate (MAR), double-labeled perimeter (dLPm), and bone formation rate (BFR)] at both skeletal sites. The bone anabolic effects of weight bearing and hPTH on dLPm and BFR at the cortical site were additive, as were the effects on MAR at the cancellous site. In contrast, weight bearing and hPTH resulted in synergistic increases in cortical bone MAR and cancellous bone dLPm and BFR. We conclude that weight bearing and PTH act cooperatively to increase bone formation by resulting in site-specific additive and synergistic increases in indexes of osteoblast number and activity, suggesting that weight-bearing exercise targeted to osteopenic skeletal sites may improve the efficacy of PTH therapy for osteoporosis.     

Gating defects of a novel Na+ channel mutant causing hypokalemic periodic paralysis

Hypokalemic periodic paralysis type 2 (hypoPP2) is an inherited skeletal muscle disorder caused by missense mutations in the SCN4A gene encoding the alpha subunit of the skeletal muscle Na+ channel (Nav1.4). All hypoPP2 mutations reported so far target an arginine residue of the voltage sensor S4 of domain II (R672/G/H/S). We identified a novel hypoPP2 mutation that neutralizes an arginine residue in DIII-S4 (R1132Q), and studied its functional consequences in HEK cells transfected with the human SCN4A cDNA. Whole-cell current recordings revealed an enhancement of both fast and slow inactivation, as well as a depolarizing shift of the activation curve. The unitary Na+ conductance remained normal in R1132Q and in R672S mutants, and cannot therefore account for the reduction of Na+ current presumed in hypoPP2. Altogether, our results provide a clear evidence for the role of R1132 in channel activation and inactivation, and confirm loss of function effects of hypoPP2 mutations leading to muscle hypoexcitability.     

Exercise training and the antioxidant alpha-lipoic acid in the treatment of insulin resistance and type 2 diabetes

One hallmark of the insulin-resistant state of prediabetes and overt type 2 diabetes is an impaired ability of insulin to activate glucose transport in skeletal muscle, due to defects in IRS-1-dependent signaling. An emerging body of evidence indicates that one potential factor in the multifactorial etiology of skeletal muscle insulin resistance is oxidative stress, an imbalance between the cellular exposure to an oxidant stress and the cellular antioxidant defenses. Exposure of skeletal muscle to an oxidant stress leads to impaired insulin signaling and subsequently to reduced glucose transport activity. Numerous studies have demonstrated that treatment of insulin-resistant animals and type 2 diabetic humans with antioxidants, including alpha-lipoic acid (ALA), is associated with improvements in skeletal muscle glucose transport activity and whole-body glucose tolerance. An additional intervention that is effective in ameliorating the skeletal muscle insulin resistance of prediabetes and type 2 diabetes is endurance exercise training. Recent investigations have demonstrated that the combination of exercise training and antioxidant treatment using ALA in an animal model of obesity-associated insulin resistance provides a unique interactive effect resulting in a greater improvement in insulin action on skeletal muscle glucose transport than either intervention individually. Moreover, this interactive effect of exercise training and ALA is due in part to improvements in IRS-1-dependent insulin signaling. These studies highlight the effectiveness of combining endurance exercise training and antioxidants in beneficially modulating the molecular defects in insulin action observed in insulin-resistant skeletal muscle.     

Molecular cloning of cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum

We have cloned and sequenced cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum. The cDNA, 16,532 base pairs in length, encodes a protein of 4,969 amino acids with a Mr of 564,711. The deduced amino acid sequence is 66% identical with that of the skeletal muscle ryanodine receptor, but analysis of predicted secondary structures and hydropathy plots suggests that the two isoforms exhibit the same topology in both transmembrane and cytoplasmic domains. A potential ATP binding domain was identified at residues 2619-2652, a potential phosphorylation site at residue 2809, and potential calmodulin binding sites at residues 2775-2807, 2877-2898, and 2998-3016. We suggest that a modulator binding domain in the protein lies between residues 2619 and 3016. Northern blot analysis of mRNA from a variety of tissues demonstrated that the cardiac isoform is expressed in heart and brain, while the skeletal muscle isoform is expressed in both fast- and slow-twitch muscle. No ryanodine receptor mRNA was detected in extracts from smooth muscle or any other non-muscle tissue examined. The two receptors are clearly the products of separate genes, and the gene encoding the cardiac muscle ryanodine receptor was localized to chromosome 1.     

Isolation and characterization of a cDNA clone encoding rat nucleoside diphosphate kinase

A cDNA clone for cytosolic nucleoside diphosphate (NDP) kinase was isolated from a cDNA library of rat skeletal muscle using synthetic oligonucleotides as probes. The clone constitutes a 621-base pair cDNA sequence including the 456-base pair coding region and 137-base pair 3'-untranslated one with polyadenylation site. The complete primary structure of NDP kinase was deduced from the coding sequence. An NH2-terminal amino acid sequence analysis suggested that the translated enzyme protein suffered proteolytic cleavage followed by modification at the alpha-NH2 group of the newly produced NH2-terminal amino acid residue. Taking this into account, it was tentatively concluded that the mature NDP kinase consists of 147 amino acid residues with a molecular weight of 16,724. Northern blot hybridization analysis showed that NDP kinase mRNA could be detected in total RNA fractions of brain, spleen, heart, lung, liver, kidney, testis as well as skeletal muscle, and that there was no difference in the size of mRNAs from these tissues. Tissue distribution of the mRNA nearly paralleled those of protein moiety and activity of the enzyme.     

Isolation and characterization of krp, a dibasic endopeptidase required for cell viability in the fission yeast Schizosaccharomyces pombe.

The activation of pro-hormones and many precursor proteins involves cleavage by endopeptidases belonging to the subtilisin-like family of enzymes. Here we describe the isolation and characterization of the first member of this family from the fission yeast Schizosaccharomyces pombe. The enzyme, which has been named krp for KEX2-related protease, is a type I membrane-bound endopeptidase that cleaves substrates after pairs of dibasic residues. It appears to be synthesized as a pre-pro-protein that is likely to undergo processing following translocation into the endoplasmic reticulum. Processing has been characterized in a cell-free translation/translocation system prepared from Xenopus eggs. Krp is N-glycosylated on all five of its potential sites and both the pre-sequence and the pro-sequence are quickly removed following translocation, the latter probably by autocatalytic cleavage. The inhibitor profile of krp broadly reflects the known properties of the eukaryotic subtilisin proteases, while its pH and Ca2+ dependence are consistent with it being active within the secretory pathway. One of its physiological substrates is likely to be the pheromone precursor pro-P-factor, which it is shown to process in an in vitro system, but identification of other substrates is complicated because, unlike other members of this family, krp is essential for cell viability.     

Functional expression of the rat heart I Na+ channel isoform. Demonstration of properties characteristic of native cardiac Na+ channels

We describe the expression of functional Na+ channels in Xenopus oocytes injected with cRNA transcribed from the rat heart I cDNA clone. The expressed rat heart I Na+ currents show kinetic properties and resistance to tetrodotoxin and saxitoxin which are characteristic of native cardiac Na+ currents. The primary amino acid sequence of the rat heart I alpha-subunit is therefore sufficient for expression of tetrodotoxin resistance, and the rat heart I clone is likely to account for the tetrodotoxin-resistant phenotype of cardiac and denervated skeletal muscle.     

A discordance in rosiglitazone mediated insulin sensitization and skeletal muscle mitochondrial content/activity in Type 2 diabetes mellitus

Skeletal muscle mitochondrial dysfunction is hypothesized to contribute to the pathophysiology of insulin resistance and Type 2 diabetes. Whether thiazolidinedione therapy enhances skeletal muscle mitochondrial function as a component of its insulin-sensitizing effect is unknown. To test this, we evaluated skeletal muscle mitochondria and exercise capacity in Type 2 diabetic subjects with otherwise normal cardiopulmonary function in response to rosiglitazone therapy. Twenty-three subjects were treated for 12 wk and underwent pre- and posttherapy metabolic stress testing and skeletal muscle biopsies. Rosiglitazone significantly ameliorated fasting glucose, insulin, and free fatty acid levels but did not augment the subjects' maximal oxygen consumption (Vo(2max)) or their skeletal muscle mitochondrial copy number. The baseline Vo(2max) correlated strongly with muscle mitochondrial copy number (r = 0.56, P = 0.018, n = 17) and inversely with the duration of diabetes (r = -0.67, P = 0.004, n = 23). Despite the global lack of effect of rosiglitazone-mediated insulin sensitization on skeletal muscle mitochondria, subjects with the most preserved functional capacity demonstrated some plasticity in their mitochondria biology as evidenced by an upregulation of electron transfer chain proteins and in citrate synthase activity. This study demonstrates that the augmentation of skeletal muscle mitochondrial electron transfer chain content and/or bioenergetics is not a prerequisite for rosiglitazone-mediated improved insulin sensitivity. Moreover, in diabetic subjects, Vo(2max) reflects the duration of diabetes and skeletal muscle mitochondrial content. It remains to be determined whether longer-term insulin sensitization therapy with rosiglitazone will augment skeletal muscle mitochondrial bioenergetics in those diabetic subjects with relatively preserved basal aerobic capacity.     

Cloning and characterization of a new isoform of skeletal muscle triadin

We have shown that several isoforms of triadin, a protein involved in calcium release process through the ryanodine receptor, are expressed in rat skeletal muscle, and we have cloned two of these isoforms. One is the rat homolog of the 95-kDa triadin identified in rabbit skeletal muscle, and the second one, shorter, is a truncated form of the previous one, but with a new unique COOH-terminal end. We propose to name the two proteins identified here Trisk 95 and Trisk 51. We have produced antibodies specific to each isoform. Using these antibodies, we have shown that the newly identified protein, Trisk 51, is actually expressed in adult rat skeletal muscle and also in rat embryo skeletal muscle. Immunofluorescent labeling of rat skeletal muscle with anti-Trisk 95, anti-Trisk 51, or anti-ryanodine receptor antibodies shows a similar localization of these proteins, in the tissue. Transfection of L6 cells with cDNA of Trisk 51 or Trisk 95 leads to the expression of proteins with the expected molecular weight, identical to those detected in rat skeletal muscle. Both proteins appear during differentiation of satellite cells in myotubes which may indicate the involvement of these two isoforms in the building of a functional calcium release machinery.