Expression of a novel recombinant stem cell factor/macrophage colony-stimulating factor fusion protein in baculovirus-infected insect cells

A novel human stem cell factor (SCF)/macrophage colony-stimulating factor (M-CSF) fusion protein gene was constructed, in which the coding regions of human SCF cDNA (1-165aa) and the truncated M-CSF cDNA (1-149aa) were connected by a linker sequence encoding a short peptide GGGGSGGGGSGG. The SCF/M-CSF gene was cloned into baculovirus transfer vector pVL1392 under the control of polyhedrin promoter and expressed in the Sf9 cells (Spodoptera frugiperda). SDS-PAGE and Western blot analysis showed that the purified fusion protein was a homodimer with a molecular weight about 84kDa under non-reducing conditions or a monomer about 42kDa under reducing conditions. The specific activity of rhSCF/M-CSF was 17 times as high as that of monomeric rhSCF to stimulate the proliferation of TF-1 cell. The results of macrophages colony-forming (CFU-M) assay performed with human bone marrow mononuclear cells demonstrated that rhSCF/M-CSF was more potent in promoting CFU-M than the equimolar of SCF, M-CSF or that of two cytokines mixture.     

Expression, purification, and characterization of a novel recombinant fusion protein, rhTPO/SCF, in Escherichia coli

Thrombopoietin (TPO) is the principal regulatory cytokine of megakaryopoiesis and thrombopoiesis and promotes all aspects of megakaryocyte development. Stem cell factor (SCF) is mainly a pleiotropic cytokine acting on hematopoiesis by promoting the survival and proliferation of hematopoietic stem cells and has a potent synergistic effect on megakaryopoiesis in the presence of TPO. Here, we report the construction, expression, and purification of a novel recombinant human thrombopoietin/stem cell factor (rhTPO/SCF) fusion protein, which consists of a truncated human thrombopoietin (1-157 a.a.) plus a truncated human stem cell factor (1-145 a.a.), linked by a peptide (GGGGSPGGSGGGGSGG). The TPO/SCF gene was cloned into the Escherichia coli expression vector pET28a and expressed in BL21(DE3) strain. The rhTPO/SCF constituted up to 6% of the total bacterial protein. Co-expression with E. coli chaperones, Trigger Factor (TF) and GroES/GroEL, and lowering cultivation temperature cooperatively improved the solubility of expressed rhTPO/SCF, resulting in about fourfold increase in the yield soluble rhTPO/SCF. The rhTPO/SCF was purified to homogeneity using anion exchange followed by metal affinity chromatography. Western blot analysis confirmed the identity of the purified protein. rhTPO/SCF stimulated a dose-dependent cell proliferation in both TF1 and Mo7e cell lines.     

Expression,refolding, and characterization of recombinant thrombopoietin/stem cell factor fusion protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Thrombopoietin/stem cell factor (TPO/SCF) is a novel fusion protein that combines the complementary biological effects of TPO and SCF into a single molecule. In this study, TPO/SCF gene was cloned into pET32a and expressed as a thioredoxin (Trx) fusion protein with a C-terminal 6His-tag in Escherichia coli BL21(DE3) under the control of T7 promoter. Trx-TPO/SCF protein approximately accounted for 20% of the total bacterial proteins and was found to accumulate in inclusion bodies. Inclusion bodies were separated from cellular debris, washed with buffer containing 2 M urea, and solubilized with 8 M urea. The refolding of Trx-TPO/SCF was then carried out by an on-column method. Soluble Trx-TPO/SCF was characterized for its dose-dependent effects on promoting cells proliferation in both TF1 and Mo7e cell lines. rhTPO/SCF was released by thrombin digestion and further purified by Ni²⁺ affinity chromatography. Western blot analysis confirmed the identities of Trx-TPO/SCF and rhTPO/SCF.     

CNTO 530: molecular pharmacology in human UT-7EPO cells and pharmacokinetics and pharmacodynamics in mice

CNTO 530 is a 58 kD antibody Fc domain fusion protein, created using Centocor's MIMETIBODY platform, that contains two EMP1 sequences as a pharmacophore. CNTO 530 has no sequence homology with EPO but acts as a novel erythropoietin receptor agonist. In UT-7(EPO) cells, CNTO 530 caused protein phosporylation of the erythropoietin receptor associated signaling pathway (Jak2, STAT5, AKT and ERK1/2). CNTO 530 also rescued these cells from apoptosis and mediated proliferation. In mice, pharmacokinetic analysis showed that CNTO 530 was slowly cleared from circulation with a t(1/2) approximately 40 h. Pharmacodynamic analysis in mice showed that a single sc dose of CNTO 530 caused a long-lived stimulation of erythropoiesis that translated into increases in red blood cell counts and hemoglobin values that were maintained for at least 28 d. In conclusion, CNTO 530 is a long-lived EPO-R agonist that stimulates erythropoiesis in a manner similar to epoetin-alpha. These data suggest that CNTO 530 may be an effective treatment of anemia in humans.     

Monkey erythropoietin gene: cloning, expression and comparison with the human erythropoietin gene

The erythropoietin (Epo) gene from Cynomolgus monkeys has been isolated from a kidney cDNA library using mixed 20-mer oligodeoxynucleotide probes. The gene encodes a 168 amino acid (aa) mature protein with a calculated Mr of 18,490 and a presumptive signal peptide of 24 aa. The Epo gene, when transfected into Chinese hamster ovary (CHO) cells, produces a glycosylated protein with an apparent Mr of 34,000. The expressed product is biologically active in vivo. The monkey gene exhibits 92% and 94% homology to the human gene at the aa and nucleotide sequence levels, respectively. When compared with the human Epo, monkey Epo has an additional 3-aa residue at the N terminus of the mature protein and a deletion of an internal lysine residue.     

Expression of a novel recombinant dual human stem cell factor in insect cells

Stem cell factor (SCF) is a hematopoietic cytokine that promotes the survival, proliferation, and differentiation of hematopoietic cells. A dual human stem cell factor (dhSCF) cDNA was constructed, which consisted of a full-length human stem cell factor cDNA plus a truncated hSCF cDNA (1-145aa), linked by a peptide (GGGGSGGGGSGG) coding region. The dhSCF gene was cloned into baculovirus transfer vector pAcSecG2T under the control of polyhedrin promoter. The Sf9 cells infected with the recombinant virus expressed rdhSCF up to 6000 U/10(6) cell in flask and 8300 U/10(6) cell in spinner flask. The rdhSCF was purified by two-step chromatography. The molecular mass of rdhSCF was examined by western blotting and HPLC analysis. The specific activity of rdhSCF was up to 3.1x10(6) U/mg, about 8.7 times as high as that of monomer rhSCF from Escherichia coli.     

Efficient expression,purification and characterization of hepatitis B virus preS1 protein from Escherichia coli

Summary The complete (encoding 119 aminon acids, aa) or partial (encoding the N-terminal 90 aa) preS1 region gene of hepatitis B virus (HBV) was fused to the 3-end of glutathione-S-transferase (GST) gene and expressed at 37 °C under the control of the inducible tac promoter in E. coli. The results showed that the fusion protein with the full length of preS1 was moderately expressed, about 10% of total cellular proteins, while the protein with the partial preS1 was highly expressed, about 33% of total cellular proteins but the half was degraded into the protein with about N-terminal 60 aa of preS1. Accordingly, GST fusion protein containing the N-terminal 56 aa of the preS1, which still encodes B-and T-cell epitopes and a hepatocyte receptor binding site, was expressed under the same induction conditions and was shown to be highly and stably expressed, about 37% of total cellular proteins. The fusion protein with the full length or N-terminal 56 aa of preS1 and the peptides were simply and successfully purified by affinity chromatography and were demonstrated to exhibit the antigenicity and immunogenicity of the preS1 antigen.     

Cloning of cDNAs encoding human interphotoreceptor retinoid-binding protein (IRBP) and comparison with bovine IRBP sequences

We have determined the sequence of the human interphotoreceptor retinoid-binding protein mRNA from three separately isolated cDNAs. The sequence is 4.28 kb long and encodes a protein of 1247 amino acids (aa) including a putative signal peptide and propeptide. The sequence is shorter (by about 1.67 kb) than the bovine mRNA with the major difference in the lengths located in the 3'-untranslated region. We suggest that this resulted from an insertion in the bovine gene or a large deletion from the human gene. The insertion/deletion is flanked on either side by sequences that are similar in the bovine and human sequences. Like the bovine polypeptide, the deduced protein sequence from the human cDNA contains a fourfold repeat, with each repeat containing about 300 aa. Among the four repeats, the identity is about 30-40%. The identity between the complete bovine and human polypeptide sequences is 84%. The identity between the nucleotide sequences is 83% (excluding the major insertion/deletion). Comparison with the bovine gene indicates that the human sequence may lack about 5-10 bp at the 5' end of the cDNA; it, however, includes a poly(A) tail at the 3' end. Thus, the human sequence is virtually full length, is similar to the bovine sequence, and contains a striking fourfold repeat.     

Cloning of human lysozyme gene and expression in the yeast Saccharomyces cerevisiae

cDNA clones encoding human lysozyme were isolated from a human histiocytic cell line (U-937) and a human placenta cDNA library. The clones, ranging in size from 0.5 to 0.75 kb, were identified by direct hybridization with synthetic oligodeoxynucleotides. The nucleotide sequence coding for the entire protein was determined. The derived amino acid sequence has 100% homology with the published amino acid (aa) sequence; the leader sequence codes for 18 aa. Expression and secretion of human lysozyme in Saccharomyces cerevisiae was achieved by placing the cloned cDNA under the control of a yeast gene promoter (ADH1) and the alpha-factor peptide leader sequence.     

Cloning of an activated human ret gene with a novel 5' sequence fused by DNA rearrangement.

By transfecting a high-Mr DNA from human stomach cancer into NIH3T3 cells, a transforming sequence that showed homology with the human ret gene was identified. The transforming sequence was found to be generated by a DNA rearrangement in the human ret proto-oncogene. This rearrangement was suggested to have occurred during the transfection procedure. The nucleotide sequences of cDNAs of the rearranged ret gene and deduced amino acid (aa) sequences revealed that the rearrangement had resulted in recombination of the 3' segment of the ret proto-oncogene with a segment of an unknown human sequence, and that the recombination had generated a novel gene encoding a fusion protein of 435 aa. The rearrangement was presumed to be responsible for activation of the ret gene.     

Expression and purification of biologically active recombinant quail stem cell factor in E. coli

Stem cell factor (SCF) is a multifunctional cytokine involved in hematopoiesis, melanogenesis and gametogenesis. Previous studies have demonstrated that avian SCF is a requirement for the proliferation and survival of various cell types in vivo and in vitro. In the current study, recombinant quail stem cell factor was produced in Escherichia coli using a prokaryotic expression system. SCF was expressed as a fusion protein with a histidine hexamer tag at the N-terminal end of the protein. Following expression, the protein was purified by affinity chromatography on the Ni-NTA column. The uninduced and induced protein lysates and the purified protein were separated by SDS-PAGE and transferred onto nitrocellulose membrane. Western blot analysis with the monoclonal antibody to the histidine tag identified SCF in the induced cell lysates and the purified sample. The recombinant SCF was approximately 22-23 kD in size. This protein was generated devoid of the signal peptide, the transmembrane domain, and the intracellular domain and, hence, resembles the soluble form of SCF. Biological activity was assayed using the in vitro survival of E12 chicken dorsal root ganglion-derived sensory neurons. The addition of recombinant quail SCF improved neuronal survival. Survival (20.6%) was the highest at the 50 ng/ml concentration of SCF. The availability of quail SCF will be a valuable tool to further resolve the function of stem cell factor in birds.     

Mapping of epitopes on the La(SS-B) autoantigen of primary Sj?gren's syndrome: identification of a cross-reactive epitope.

Autoepitopes on the ribonucleoprotein La(SS-B) were identified by using recombinant La(SS-B) polypeptides and sera from 166 patients with the antinuclear autoantibody anti-La(SS-B). The La(SS-B) polypeptides were encoded by polymerase chain reaction-derived overlapping or nonoverlapping fragments of the La(SS-B) gene, which encodes a protein of 408 amino acids (aa). Of the 166 sera tested, 99% reacted with a fusion protein comprising the first 107 N-terminal aa (LaA); 91% reacted with a fusion protein comprising aa 111 to 242 (LaC), and 91% reacted with a fusion protein comprising aa 346 to 408 (LaL2/3) at the C terminus of La(SS-B). The order of immunodominance as assessed by the number of sera reacting with each epitope and the strength of the reactivity was LaA (aa 1 to 107) greater than LaC (aa) 111 to 242) much greater than LaL2/3 (aa 346 to 408). Cross-reactivity was observed between antibodies eluted from LaC (aa 111 to 242) and LaL2/3 (aa 346 to 408), but there was no significant primary sequence homology between the two regions. The LaC region contained at least two epitopes, one encompassing a putative RNA-binding motif (aa 112 to 187) which was recognized by 83% of patient sera. Serial serum samples from three patients showed that the antibody response to La(SS-B) was initially directed to the N terminus (LaA, aa 1 to 107), but over a period of time all three major epitopes, including that encompassing the putative RNA-binding motif, were recognized. This result suggests that the primary immune response to La(SS-B) is restricted to an immunodominant epitope. As the specificity of the autoantibody response broadens, it includes the RNA-binding motif, which may have important implications for the expression of disease.     

Efficient expression and characterization of hepatitis B virus preS2 antigen in Escherichia coli

The complete (encoding 55 amino acids, aa) or partial (encoding aa 1–26) preS2 region gene of hepatitis B virus (HBV) was fused to the 3-end of glutathion-S-transferase (GST) gene and expressed under the control of the inducible tac promoter in Escherichia coli at 37 °C. The fusion protein with the complete preS2 region was moderately expressed (8%) while the protein with the N-terminal 26 aa was expressed at a higher level, yielding about 20% of the total cellular proteins. The GST-preS2 (aa 1–26) protein, which contains the immunodominant epitope, was produced form the soluble protein fraction of the recombinant bacteria and purified by affinity chromatography using glutathione-agarose column. The purified preS2 fusion protein showed the antigenicity of preS2, as assessed by indirect and competitive ELISAs.     

rhEPO-L-Fc融合蛋白的表达、生物活性和初步药动学分析

为了延长人促红细胞生成素(hEPO)体内半衰期以达到更好的药效,制备通过柔性接头相连接的重组人红细胞生成素-IgG1 Fc融合蛋白(rhEPO-L-Fc),并对其生物学活性和体内药动学进行初步研究.利用PCR技术构建rhEPO-L-Fc融合基因,克隆至表达载体pOptiVEC-TOPO ,在二氢叶酸还原酶缺陷型中国仓鼠卵巢细胞(CHO-dhfr-)表达.Protein A亲合层析柱纯化融合蛋白,SDS-PAGE、质谱、Western blotting鉴定表达产物,细胞增殖实验检测融合蛋白的体外活性,动物实验检测融合蛋白的体内活性和半衰期.成功构建pOptiVEC-TOPO -rhEPO-L-Fc重组子,实现了在CHO细胞表达,纯化后的rhEPO-L-Fc融合蛋白经鉴定,其分子量和特异性均与理论值相符,能刺激体外培养的EPO依赖型细胞生长,ED50为2 ng/mL,且明显增加大鼠外周血网织红细胞数,体内消除半衰期达到27 h.rhEPO-L-Fc融合蛋白能延长hEPO体内半衰期,为其临床研究奠定了基础.     

Dendritic cell-associated lectin-1: a novel dendritic cell-associated,C-type lectin-like molecule enhances T cell secretion of IL-4

We have characterized dendritic cell (DC)-associated lectin-1 (DCAL-1), a novel, type II, transmembrane, C-type lectin-like protein. DCAL-1 has restricted expression in hemopoietic cells, in particular, DCs and B cells, but T cells and monocytes do not express it. The DCAL-1 locus is within a cluster of C-type lectin-like loci on human chromosome 12p12-13 just 3' to the CD69 locus. The consensus sequence of the DCAL-1 gene was confirmed by RACE-PCR; however, based on sequence alignment with genomic DNA and with various human expressed sequence tags, we predict that DCAL-1 has two splice variants. C-type lectins share a common sequence motif of 14 invariable and 18 highly conserved aa residues known as the carbohydrate recognition domain. DCAL-1, however, is missing three of the cysteine residues required to form the standard carbohydrate recognition domain. DCAL-1 mRNA and protein expression are increased upon the differentiation of monocytes to CD1a(+) DCs. B cells also express high levels of DCAL-1 on their cell surface. Using a DCAL-1 fusion protein we identified a population of CD4(+) CD45RA(+) T cells that express DCAL-1 ligand. Coincubation with soluble DCAL-1 enhanced the proliferation of CD4(+) T cells in response to CD3 ligation and significantly increased IL-4 secretion. In contrast, coincubation with soluble DC-specific ICAM-3-grabbing nonintegrin (CD209) fusion protein as a control had no effect on CD4(+) T cell proliferation or IL-4 and IFN-gamma secretion. Therefore, the function of DCAL-1 on DCs and B cells may act as a T cell costimulatory molecule, which skews CD4(+) T cells toward a Th2 response by enhancing their secretion of IL-4.     

Expression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner

Stem cell factor (SCF) known as the c-kit ligand, plays important roles in spermatogenesis, melanogenesis and early stages of hematopoiesis. As for the latter, SCF is essential for growth and expansion of hematopoietic stem and progenitor cells. We herein describe the production of recombinant murine SCF from Escherichia coli as soluble thioredoxin-fusion protein. The formation of insoluble and inactive inclusion bodies, usually observed when SCF is expressed in E. coli, was almost entirely prevented. After purification based on membrane adsorber technology, the fusion protein was subsequently cleaved by TEV protease in order to release mature mSCF. Following dialysis and a final purification step, the target protein was isolated in high purity. Bioactivity of mSCF was proven by different tests (MTT analogous assay, long-term proliferation assay) applying a human megakaryocytic cell line. Furthermore, the biological activity of the uncleaved fusion protein was tested as well. We observed a significant activity, even though it was less than the activity displayed by the purified mSCF. In summary, avoiding inclusion body formation we present an efficient production procedure for mSCF, one of the most important stem cell cytokines.     

Nucleotide sequence of the Synechococcus sp. PCC7942 branching enzyme gene (glgB): expression in Bacillus subtilis

The nucleotide sequence of the Synechococcus sp. PCC7942 glgB gene has been determined. The gene contains a single open reading frame (ORF) of 2322 bp encoding a polypeptide of 774 amino acids (aa) with an Mr of 89,206. Extensive sequence similarity exists between the deduced aa sequence of the Synechococcus sp. glgB gene product and that of the Escherichia coli branching enzyme in the middle portions of the proteins (62% identical aa). In contrast, the N-terminal portions shared little homology. The sequenced region which follows glgB contains an ORF encoding 79 aa of the N terminus of a polypeptide that shares extensive sequence similarity (41% identical aa) with human and rat uroporphyrinogen decarboxylase. This suggests that the region downstream from glgB contains the hemE gene and, therefore, that the organization of genes involved in glycogen biosynthesis in Synechococcus sp. is different from that described for E. coli. A fusion gene was constructed between the 5' end of the Bacillus licheniformis penP gene and the Synechococcus sp. glgB gene. The fusion gene was efficiently expressed in the Gram+ micro-organism Bacillus subtilis and specified a branching enzyme with an optimal temperature for activity similar to the wild-type enzyme.