Thrombin and H64A subtilisin cleavage of fusion proteins for preparation of human recombinant parathyroid hormone

Human parathyroid hormone, hPTH, an 84 amino acid polypeptide, was produced intracellularly inEscherichia coli as a fusion protein, linked to the C-terminus of a 15 kD IgG-binding protein. Approximately 100 mg fusion protein was obtained per liter fermentation medium. To test the efficiency of two alternative enzymatic cleavage methods, two fusion proteins differing only in the linker region were constructed. Cleavage of a Phe-Phe-Pro-Arg linker was obtained with bovine thrombin and cleavage of a Phe-Ala-His-Tyr linker with recombinant H64A subtilisin. Both enzmes yielded the correct N-terminus and cleaved their respective linkers quantitatively, although additional internal cleavage sites in hPTH were detected and characterized. The linker cleavage conditions were optimized and hPTH was purified to homogeneity. Thrombin cleavage resulted in a final yield of 5 mg hPTH/L, while H64A subtilisin cleavage was more specific and gave 8 mg/L. The purified recombinant product was identical to native hPTH and exhibited full biological activity in an adenylate cyclase assay.     

High-yield expression of fully bioactive N-terminal parathyroid hormone analog in Escherichia coli

A fully active analog of human parathyroid hormone (hPTH) has been produced by recombinant expression in Escherichia coli. Initially, a nucleotide sequence encoding hPTH(1-34)-Asp-Pro was ligated to a proinsulin gene in the plasmid pUC8, for the eventual expression of a fusion protein of 137 amino acids. Unexpectedly, the proinsulin gene and 340 bp downstream were deleted by an unknown mechanism during transformation of the E. coli. This resulted in a new plasmid encoding a small (72-amino acid) fusion product of hPTH(1-34)-Asp35-Pro36-X, where X is a 36-residue "arbitrary" downstream sequence of pUC8. The fusion product was efficiently expressed and the hPTH analog, [Asp35]hPTH-(1-35), was readily released by acid cleavage, with a yield of 100 mg/L. This analog had an effective concentration for half-maximal adenylyl cyclase stimulation (EC50) in rat osteosarcoma cells of 14 nM, which was identical to that for hPTH-(1-34). In the ovariectomized rat model of osteoporosis, [Asp35]hPTH-(1-35) was fully active as a bone anabolic agent.     

Large scale preparation of recombinant human parathyroid hormone 1-84 from Escherichia coli

Human parathyroid hormone (hPTH) is a promising agent in the treatment of osteoporosis. The intact recombinant human parathyroid hormone [rhPTH(1-84)] was prepared in a large scale from Escherichia coli using a soluble fusion protein strategy. With degenerate codons, gene of hPTH(1-84) was synthesized, ligated with pET32a(+) vector, and then expressed in E. coli BL21(DE3) cells. The soluble fusion protein His(6)-thioredoxin-hPTH(1-84) was harvested after purification by immobilized metal affinity chromatography (IMAC). Following enterokinase cleavage, ion-exchange-chromatography (IEC) and size-exclusive-chromatography (SEC) were used, and finally, over 300mg/l intact hPTH(1-84) with high purity up to 99% was obtained. The purified rhPTH(1-84) was confirmed by mass spectrometry and N-terminal/C-terminal amino-acid sequence analysis. Additionally, this product stimulated adenylate cyclase in Rat Osteosarcoma Cell UMR-106 at the same extent as hPTH standards, indicating that the purified rhPTH(1-84) has full biological activity. The efficient procedure for expression and purification of rhPTH(1-84) may be useful for the mass production of this important protein.     

High-Level Production of Recombinant Human Parathyroid Hormone 1-34

Expression of the synthetic human parathyroid hormone 1-34 [hPTH(1-34)] gene by a gene fusion strategy was demonstrated. hPTH(1-34) was produced at the C terminus of the partner peptides involving amino acids 1 to 97, 1 to 117, or 1 to 139 of a modified Escherichia coli β-galactosidase by linker peptides containing oligohistidine of different lengths. The fusion proteins in the inclusion bodies were rendered soluble with urea and subjected to site-specific cleavage with the secretory type yeast Kex2 protease. Optimal expression and enzymatic processing were achieved in the fusion protein βG-117S4HPT, constructed from amino acids 1 to 117 of β-galactosidase and the linker of HHHHPGGSVKKR. The fusion protein accumulated more than 20% of the E. coli total protein. The hPTH(1-34) was purified up to 99.5% with a good yield of 0.5 g/liter of culture. The purified product was identified as intact hPTH(1-34) by amino acid analysis and N-terminal sequencing.     

Human parathyroid hormone related protein fragment-(1-34) had glucose-6-phosphate dehydrogenase activity on distal convoluted tubules in cytochemical bioassay

In the present study, the action of parathyroid hormone related protein (PTHrP) on glucose-6-phosphate dehydrogenase (G6PD) activity of the distal convoluted tubules was examined utilizing cytochemical bioassay (CBA). Recently full amino acid residues of human PTHrP (hPTHrP), one of the causative agents of HHM, was identified based on the cDNA clone using BEN cells. We synthesized hPTHrP-(1-34) and examined the effect of this protein on G6PD activities on the distal convoluted tubules, and compared its bioactivity to that of human parathyroid hormone (hPTH)-(1-84). hPTHrP-(1-34) stimulated G6PD activity in a log linear fashion with equivalent activity to that of hPTH-(1-84) on a molar basis. Conclusively, we found that PTHrP act on distal convoluted tubules similar to hPTH.     

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.     

BPTI and N-terminal extended analogues generated by factor Xa cleavage and cathepsin C trimming of a fusion protein expressed in Escherichia coli

A recombinant gene for BPTI (bovine pancreatic trypsin inhibitor) is expressed in Escherichia coli using a MBP (maltose-binding protein) fusion vector. BPTI is fused through an FXa (blood coagulation factor Xa protease) target sequence (Ile-Glu-Gly-Arg) to the C-terminus of MBP. The MBP moiety of the hybrid protein enables purification in one step utilizing MBP's affinity to cross-linked amylose, and the FXa target sequence allows specific cleavage of the hybrid protein. Effective FXa cleavage is achieved by spacing the FXa target sequence and Arg-1 of the BPTI sequence with four residues (Met-Glu-Ala-Glu). The resulting N-terminal extended BPTI is readily converted to the wild-type sequence by trimming with cathepsin C exopeptidase, for the activity of which the spacing tetrapeptide is optimized. FXa cleavage is prohibited when the target sequence is placed next to Arg-1. In this construction, off-target cleavage at a somewhat homologous sequence (Val-Pro-Gly-Arg) results in five- or six-residue extended BPTI, indicating new details of the FXa specificity. The yield of highly purified recombinant BPTI is 3-6 mg/liter of culture, making the MBP-BPTI expression system convenient for the production of sufficient amounts of protein for NMR studies. 1H NMR is used to analyze the N-extended BPTI analogues.     

Characterization of a K26Q site-directed mutant of human parathyroid hormone expressed in yeast

Human parathyroid hormone (hPTH) is susceptible to proteolytical cleavage both in humans and when expressed as a secretory product in Escherichia coli (H?gseth, A., Blingsmo, O. R., Saether, O., Gautvik, V. T., Holmgren, E., Hartmanis, M., Josephson, S., Gabrielsen, O. S., Gordeladze, J. O., Alestr?m, P., and Gautvik, K. M. (1990) J. Biol. Chem. 265, 7338-7344) and Saccharomyces cerevisiae (Gabrielsen, O. S., Reppe, S., Saether, O., Blingsmo, O. R., Sletten, K., Gordeladze, J. O., H?gset, A., Gautvik, V. T., Alestr?m, P., Oyen, T. B., and Gautvik, K. M. (1990) Gene (Amst.) 90, 255-262). In the latter system, one major site of cleavage was identified (Arg25-Lys26 decreased Lys27). To produce hPTH resistant to this proteolytic processing, a point mutation changing Lys26 to Gln was introduced, and the modified gene expressed in S. cerevisiae as a fusion protein with the alpha-factor leader sequence. The resulting major form of hPTH secreted to the growth medium was of full length showing that the mutation had eliminated internal processing. Consequently, the yield of the mutant hormone was significantly higher than obtained with the natural peptide. Using improved purification procedures, a significantly higher purity was also obtained. The secreted mutant hPTH-(1-84,Q26) had the correct size, full immunological reactivity with two different hPTH antisera, correct amino acid composition and N-terminal sequence, and correct mass as determined by mass spectrometry. Furthermore, the introduced mutation did not reduce the biological activity of the hormone as judged from its action in three biological assay systems: 1) a hormone-sensitive osteoblast adenylate cyclase assay; 2) an in vivo calcium mobilizing assay in rats; and 3) an in vitro bone resorption assay.     

Studies on the production of human parathyroid hormone by recombinant Escherichia coli

Expression of human parathyroid hormone, hPTH(-1-84), by Escherichia coli N4830: pEX-PPTH was studied in controlled bioreactors. The hPTH is expressed as a fusion protein under control of the bacteriophage p_R promoter. In batch runs, low biomass concentrations but high specific hPTH productivities were obtained with complex TY (bactotryptone and yeast extract) medium whereas high biomass concentration and low specific productivities were found when fructose was used instead of bactotryptone (YF medium). The preinduction temperature was always 30°C; the temperature shift to induce production of fusion protein was varied from 36 to 42°C. Formation of hPTH passed a pronounced maximum as a function of induction temperature when using YF medium. However, the optimum temperature shift was 38°C for both media used. For this temperature increase both media yielded about the same volumetric hPTH productivity (approx. 30 mg hPTH/l per hour). By applying a fedbatch strategy for the YF medium, the productivity of the recombinant protein could be further increased more than fourfold. Compared to shake-flask experiments, the hPTH yield could be increased by a factor larger than 20.     

Overexpression in Escherichia coli and purification of human fibroblast growth factor (FGF-2)

Basic fibroblast growth factor (FGF-2) is a member of a large family of structurally related proteins that affect the growth, differentiation, migration, and survival of many cell types. The human FGF-2 gene (encoding residues 1–155) was synthesized by PCR from 20 oligonucleotides and cloned into plasmid pET-32a. A high expression level (1 g/liter) of a fused protein thioredoxin/FGF-2 was achieved in Escherichia coli strain BL21(DE3). The fusion protein was purified from the soluble fraction of cytoplasmic proteins on a Ni-NTA agarose column. After cleavage of the thioredoxin/FGF-2 fusion with recombinant human enteropeptidase light chain, the target protein FGF-2 was purified on a heparin-Sepharose column. The yield of FGF-2 without N- and C-terminal tags and with high activity was 100 mg per liter of cell culture. Mutations C78S and C96S in the amino acid sequence of the protein decreased FGF-2 dimer formation without affecting its solubility and biological activity.     

Cloning, expression and purification of the luminal domain of spinach photosystem 1 subunit PsaF functional in binding to plastocyanin and with a disulfide bridge required for folding

The photosystem 1 subunit PsaF is involved in the docking of the electron-donor proteins plastocyanin and cytochrome c? in eukaryotic photosynthetic organisms. Here we report the expression, purification and basic characterization of the luminal domain of spinach PsaF, encompassing amino-acid residues 1-79. The recombinant protein was expressed in Escherichia coli BL21 (DE3) using a pET32 Xa/LIC thioredoxin fusion system. The thioredoxin fusion protein contained a His? tag and was removed and separated from PsaF through proteolytic digestion by factor Xa followed by immobilized metal affinity chromatography. Further purification with size-exclusion chromatography resulted in a final yield of approximately 6 mg PsaF from one liter growth medium. The correct identity after the factor Xa treatment of PsaF was verified by FT-ICR mass spectrometry which also showed that the purified protein contains an intact disulfide bridge between Cys residues 6 and 38. Secondary structure and folding was further explored using far-UV CD spectroscopy indicating a α-helical content in agreement with the 3.3 ?-resolution crystal structure of photosystem I. and a helix-coil transition temperature of 29 °C. Thermofluorescence studies showed that the disulfide bridge is necessary to keep the overall fold of the protein and that hydrophobic regions become exposed at 50-65 °C depending on the ionic strength. The described expression and purification procedure can be used for isotopic labeling of the protein and 1?N-HSQC NMR studies indicated a slow or intermediate exchange between different conformations of the prepared protein and that it belongs to the molten-globule structural family. Finally, by using a carboxyl- and amine-reactive zero-length crosslinker, we have shown that the recombinant protein binds to plastocyanin by a specific, native-like, electrostatic interaction, hence, confirming its functionality.     

A membrane-bound metallo-endopeptidase from rat kidney hydrolyzing parathyroid hormone. Purification and characterization

A metallo-endopeptidase, which appears to be an integral membrane protein of rat kidney, was purified to homogeneity by a series of standard chromatographic procedures. This enzyme significantly hydrolyzed human parathyroid hormone [hPTH(1-84)] and a synthetic substrate Suc-Leu-Leu-Val-Tyr-Mec (Suc = succinyl, Mec = 4-methyl-coumarinyl-7-amide). The purified enzyme had apparent molecular masses of 250 kDa on gel filtration, and 88 kDa and 245 kDa on sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions, respectively. Its pH optimum for activity was 8.0-8.5 and its isoelectric point was pH 4.9. Its activity was inhibited by EDTA, EGTA and o-phenanthroline, but not by phosphoramidon. The metal-depleted enzyme was reactivated by the addition of metal ions. The enzyme was also inhibited by chymostatin and eglin C, and by thiol compounds. Of the synthetic substrates examined, the enzyme hydrolyzed only Suc-Leu-Leu-Val-Tyr-Mec, one of the synthetic substrates for alpha-chymotrypsin. It did not hydrolyze synthetic substrates with less than four amino acid residues with tyrosine in the P1 position. The enzyme hydrolyzed hPTH and reduced hen egg lysozyme but did not hydrolyze azocasein or [3H]methyl-casein. NH2-terminal amino acid sequence analyses of the degradation products of hPTH(1-84) and reduced hen egg lysozyme by the purified enzyme revealed that the enzyme preferentially cleaved these peptides at peptide bonds flanked by hydrophilic amino acid residues. Amino acid analyses showed that the main degradation products of PTH were hPTH(17-29), hPTH(30-38) and hPTH(74-84). The ability of the enzyme to hydrolyze peptide bonds flanked by hydrophilic amino acid residues and its inability to degrade azocasein distinguish it from several other kidney endopeptidases reported, such as endopeptidase 24.11 and meprin.     

Amino-terminal modifications of human parathyroid hormone (PTH) selectively alter phospholipase C signaling via the type 1 PTH receptor: implications for design of signal-specific PTH ligands.

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) activate the PTH/PTHrP receptor to trigger parallel increases in adenylyl cyclase (AC) and phospholipase C (PLC). The amino (N)-terminal region of PTH-(1-34) is essential for AC activation. Ligand domains required for activation of PLC, PKC, and other effectors have been less well-defined, although some studies in rodent systems have identified a core region [hPTH-(29-32)] involved in PKC activation. To determine the critical ligand domain(s) for PLC activation, a series of truncated hPTH-(1-34) analogues were assessed using LLC-PK1 cells that stably express abundant transfected human or rat PTH/PTHrP receptors. Phospholipase C signaling and ligand-binding affinity were reduced by carboxyl (C)-terminal truncation of hPTH-(1-34) but were coordinately restored when a binding-enhancing substitution (Glu(19) --> Arg(19)) was placed within hPTH-(1-28), the shortest hPTH peptide that could fully activate both AC and PLC. Phospholipase C, but not AC, activity was reduced by substituting Gly(1) for Ser(1) in hPTH-(1-34) and was eliminated entirely by removing either residue 1 or the alpha-amino group alone. These changes did not alter binding affinity. These findings led to design of an analogue, [Gly(1),Arg(19)]hPTH-(1-28), that was markedly signal-selective, with full AC but no PLC activity. Thus, the extreme N-terminus of hPTH constitutes a critical activation domain for coupling to PLC. The C-terminal region, especially hPTH-(28-31), contributes to PLC activation through effects upon receptor binding but is not required for full PLC activation. The N-terminal determinants of AC and PLC activation in hPTH-(1-34) overlap but are not identical, as subtle modifications in this region may dissociate activation of these two effectors. The [Gly(1),Arg(19)]hPTH-(1-28) analogue, in particular, should prove useful in dissociating AC- from PLC-dependent actions of PTH.     

High-Level Production of Human Parathyroid Hormone (hPTH) by Induced Expression inSaccharomyces cerevisiae

Saccharomyces cerevisiaewas used as host for high-level production of intact human parathyroid hormone (hPTH). The yield increased about 30-fold by changing from the constitutive MFα promoter to the inducibleCUP1promoter in the expression cassettes, use of another host strain, and optimization of growth conditions where especially the pH value was crucial. The secreted products consisted mainly of intact hormone, hPTH(1-84). In addition, two C-terminally truncated forms that lacked the four or five last amino acid residues, hPTH(1-80) and hPTH(1-79), were identified. These hPTH forms migrated aberrantly by SDS–PAGE as 14-kDa proteins, while the real masses measured by mass spectrometry on HPLC-purified products were about 9 kDa. Availability of such easily purified truncated forms will be valuable for studies of how the C-terminal residues affect the structure and function of the hormone. Combination of mutations and disruptions of the host genes encoding proteinase A, B, carboxypeptidase Y, and Kex1p or Mkc7p did not influence the C-terminal deletions. The secretion of hPTH could be enhanced by overexpression of the yeast syntaxin geneSSO2, but the total level of the hormone was not improved due to impaired growth.     

Engineering of a Pichia pastoris expression system for secretion of high amounts of intact human parathyroid hormone

Human parathyroid hormone (hPTH) is involved in calcium metabolism, and the unique ability of this hormone to stimulate bone growth makes it a promising agent in the treatment of osteoporosis. We have engineered the methylotrophic yeast Pichia pastoris for the production of over 300 mg intact hPTH per liter growth medium. The presence of 10 mM EDTA in the culture medium was essential to obtain this high hormone yield, indicating that metallopeptidases are mainly responsible for the otherwise instability of hPTH. Furthermore, the secretion process of hPTH was considerably improved by coexpression of Saccharomyces cerevisiae protein disulphide isomerase (ScPDI). Since hPTH does not contain any cystein residues, this effect may be indirect or ascribed to the chaperone activity of PDI. Contrary to the situation in S. cerevisiae, use of a protease-deficient host strain provided no additional advantage. The hormone secreted by P. pastoris was not subjected to proteolytic processing by Kex2p in the two internal tribasic sites, nor were any C-terminal truncated hPTH forms detected. However, the P. pastoris hPTH producing transformants cosecreted ubiquitin to the culture medium, possibly as a result of a stress-related response.     

Purification and characterization of recombinant human insulin-like growth factor II (IGF-II) expressed as a secreted fusion protein in Escherichia coli

Human insulin-like growth factor II (IGF-II) was produced in an Escherichia coli ompT strain as a 22.5-kDa fusion protein. IGF-II was fused to the carboxy-terminal of a synthetic 15-kDa IgG-binding protein, originating from staphylococcal protein A, via a unique methionine linker. During fermentation, the fusion protein was exported to the growth medium at levels exceeding 900 mg/liter and subsequently affinity purified on IgG Sepharose followed by ion exchange on S Sepharose. After chemical cleavage with CNBr, yielding an authentic IGF-II molecule, the recombinant IGF-II was purified to homogeneity by a two step procedure involving ion-exchange and reverse-phase HPLC. A substantial fraction of the secreted protein was found to be biologically active, eliminating the need for complex refolding procedures. The yield of highly purified and biologically active IGF-II was 5-7 mg/liter of fermenter broth. The IGF-II produced by this method displayed biochemical, immunological, receptor binding, and biological activity properties equal to those of native IGF-II isolated from human serum.     

A PagP fusion protein system for the expression of intrinsically disordered proteins in Escherichia coli

PagP, a beta-barrel membrane protein found in Gram-negative bacteria, expresses robustly in inclusion bodies when its signal sequence is removed. We have developed a new fusion protein expression system based on PagP and demonstrated its utility in the expression of the unstructured N-terminal region of human cardiac troponin I (residues 1-71). A yield of 100mg fusion protein per liter M9 minimal media was obtained. The troponin I fragment was removed from PagP using cyanogen bromide cleavage at methionine residues followed by nickel affinity chromatography. We further demonstrate that optimal cleavage requires complete reduction of methionine residues prior to cyanogen bromide treatment, and this is effectively accomplished using potassium iodide under acidic conditions. The PagP-based fusion protein system is more effective at targeting proteins into inclusion bodies than a commercially available system that uses ketosteroid isomerase; it thus represents an important advance for producing large quantities of unfolded peptides or proteins in Escherichia coli.     

人TFF2基因的原核表达与纯化(英文)

人三叶因子2(hTFF2)具有促进细胞迁移和抑制细胞凋亡的活性,所以被认为是胃肠黏膜修复的启动者之一。因为从人组织中获得hTFF2比较困难,而且体外产生的重组hTFF2大都以融合蛋白的形式存在,所以该研究的目的是在体外产生不带任何融合蛋白的游离型hTFF2。hTFF2的开放阅读框被插入pET-32a(+)表达载体,然后在大肠杆菌中表达出带有硫氧还蛋白融合部分的hTFF2融合蛋白。进而利用融合蛋白的组氨酸标签使用镍亲和色谱柱以及反向高压色谱柱对目的蛋白进行纯化。23°C,FXa因子裂解纯度高达95%的融合蛋白以得到游离型hTFF2。在去除FXa因子和尚未被切开的融合蛋白后,获得的游离型hTFF2被SDS-PAGE和Westernblotting所证实。重组游离型hTFF2的产量约为5mg/L,并且hTFF2能促进IEC-6细胞的迁移以及体外的伤口修复,而这些活性是依赖于ERK1/2的激活。同时,hTFF2也能抑制50μmol/L神经鞘氨醇所引起的HCT-116细胞的凋亡。总之,研究结果表明,在大肠杆菌中高产量地成功表达出具有生物学活性的游离型hTFF2,这为研究TFF2的分子机制,以及研制和开发TFF2的相关药物都提供很大的帮助。     

Synthesis and characterization of extended and deleted recombinant analogues of parathyroid hormone-(1-84): correlation of peptide structure with function

Recombinant analogues of human parathyroid hormone [hPTH-(1-84)] were expressed in Escherichia coli harboring plasmids containing synthetic genes under the control of the lac promoter. The level of expression of the gene encoding the truncated analogue, hPTH-(3-84), was greater than that of the gene encoding full-length hPTH-(1-84) but less than that of the gene encoding proparathyroid hormone (hProPTH). This may be due in part to the relative efficiency of translation of the mRNA as suggested by secondary structure analysis and in part because of enhanced stability of the extended peptide. Formylmethionyl derivatives of hProPTH and of hPTH-(3-84) and underivatized hPTH-(3-84) were purified by HPLC, and their identity was confirmed by NH2-terminal sequencing and amino acid analysis. The bioactivity of these recombinant peptides was then tested in skeletal and renal adenylate cyclase assays in vitro and in assays examining effects on plasma and urine calcium and phosphate levels and on urine cyclic AMP levels in vivo. The NH2-terminally extended analogue fMet-hProPTH displayed 10% of the in vitro activity of hPTH-(1-84) and was a partial agonist in vivo. The peptides hPTH-(3-84) and fMet-hPTH-(3-84) were inert in vitro and were very weak in vitro antagonists when compared to the NH2-terminal analogue bovine [Nle8,18Tyr34]PTH-(3-34)-NH2. In vivo, hPTH-(3-84) and the bPTH-(3-34) analogue, when assayed at a 10:1 molar ratio relative to bPTH-(1-84), were each inert, and neither demonstrated antagonist activity at these concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

提高Xa因子酶切效率的策略

为提高Xa因子对融合蛋白CBD-IGF和CBD-PACAP的酶切效率 ,以便高效释放非融合的重组多肽 ,利用基因工程技术在两个融合蛋白中Xa因子识别位点 (Ile-Glu-Gly-Arg↓ )前均引入 7个氨基酸组成的富含甘氨酸柔性短肽 (Gly-Thr-Gly-Gly-Gly-Ser-Gly)。纤维素亲和层析纯化各个融合蛋白 ,比较Xa因子对引入短肽前、后融合蛋白的酶切效率。比较结果表明 :短肽的引入不同程度地提高了融合蛋白CBD-IGF和CBD-PACAP对Xa因子的敏感性 ;但总体上CBD-IGF对Xa因子的敏感性比CBD-PACAP低。此研究结果提供了一种提高Xa因子酶切效率的策略。