-
[show abstract]
[hide abstract]
ABSTRACT: Streptomyces sp. 139 was identified to produce a new exopolysaccharide Ebosin (139A) with antirheumatic arthritis activity in vivo. The Ebosin biosynthesis gene cluster(31.3 kb; GenBank Accession Number: AY131229) containing 22 ORFs (ste1-ste22) of Streptomyces sp. 139 had been reported previously. In this paper,we present experimental evidence for the identity of the ste6 gene product as a UDP-glucose dehydrogenase (UDPGDH). With pET-30a as vector,the gene was cloned and expressed in Escherichia coli BL21 (DE3). The expressed protein was purified to homogeneity by His-Bind resin affinity chromatograpy and it was able to catalyze UDP-glucose to UDP-glucuronic acid. To evaluate the function of ste6, the gene was disrupted by a single-crossover homologous recombination event and the result showed that ste6 is required in Ebosin biosynthesis.
Acta Genetica Sinica 12/2005; 32(11):1213-20.
-
[show abstract]
[hide abstract]
ABSTRACT: Proteasomes are self-compartmentalizing proteases first discovered in eukaryotes but also occurring in archaea and in bacteria belonging to the order Actinomycetales. In bacteria, proteasomes have so far no known function. In order to evaluate the influence of the 20S proteasome on the production of heterologous proteins by Streptomyces lividans TK24, the production of a number of heterologous proteins, including soluble human tumour necrosis factor receptor II (shuTNFRII) and salmon calcitonin (sCT), was compared with the wild-type TK24, a proteasome-deficient mutant designated PRO41 and a strain complemented for the disrupted proteasome genes (strain PRO41R). S. lividans cells lacking intact proteasome genes are phenotypically indistinguishable from the wild-type or the complemented strain containing functional proteasomes. Using the expression and secretion signals of the subtilisin inhibitor of Streptomyces venezuelae CBS762.70 (Vsi) for shuTNFRII and those of tyrosinase of Streptomyces antibioticus (MelC1) for the production of sCT, both proteins were secreted in significantly higher amounts in the strain PRO41 than in the wild-type S. lividans TK24 or the complemented strain PRO41R. However, the secretion of other heterologous proteins such as shuTNFRI was not enhanced in the proteasome-deficient strain. This suggests that S. lividans TK24 can degrade some heterologous proteins in a proteasome-dependent fashion. The proteasome-deficient strain may therefore be useful for the efficient production of these heterologous proteins.
Microbiology 10/2005; 151(Pt 9):3137-45. · 3.06 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To establish a new drug screening model based on transcriptional regulation of human high density lipoprotein (HDL) receptor gene CD36 and LIMPII analogous-1 (CLA-1) for discovering up-regulator of this receptor.
The upstream regulatory sequence of CLA-1 was obtained by polymerase chain reaction. A recombinant reporter plasmid pGL3-CLAP was constructed by inserting the regulatory sequence upstream of luciferase gene of pGL3-Basic. Human hepatoma cell line BEL-7402 was transfected with pGL3-CLAP. Samples were detected by testing luciferase activity of transfected BEL-7402 cells in microtiter wells.
The drug screening model was established and optimized. Significant difference was present between pGL3-CLAP and pGL3-Basic transfected BEL-7402 cells (P< 0.001), and coefficient of variation was less than 10%. After primary and secondary screening, 1 compounds and 3 fermentation extracts had up-regulating activities.
This new drug screening model may be efficiently used to screen up-regulators of human HDL receptor expression, which might become lead compounds for new anti-atherosclerosis drugs.
Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae 09/2004; 26(4):354-8.
-
[show abstract]
[hide abstract]
ABSTRACT: Interleukin-4 (IL-4) is a pleiotropic cytokine which plays a pivotal role in shaping immune responses and mediating important proinflammatory functions in asthma. This cytokine exerts its biological effects through binding to its receptor (IL-4R) complex, with the alpha chain as the high-affinity binding subunit. Soluble IL-4R (sIL-4R) lacks the transmembrane and cytoplasmic domains, so it cannot induce cellular activation. By acting as a decoy to circulating IL-4 and neutralizing its activity, the high specificity and affinity of sIL-4R make it ideal as an IL-4 antagonist. In this study, a sIL-4R cDNA encoding the extracellular domain of IL-4R alpha chain was cloned into a Streptomyces-Escherichia coli shuttle plasmid pSGLgpp and expressed secretly in Streptomyces lividans TK 24. On SDS-PAGE gel, the expressed sIL-4R protein showed a Mw of 24 kDa, agreeable with the predicted size. The N-terminal sequence of the protein was also determined, confirming its identity and indicating that no degradation occurred at the N-terminus. With DEAE-Sepharose Fast Flow and Superdex HR 75 columns, the protein was purified and used on HPLC analysis, the purity reaching about 90%. The results of the ligand-binding blot and ELISA showed that such protein has biological activity of binding with ligand IL-4.
Protein Expression and Purification 08/2004; 36(1):139-45. · 1.59 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Interleukin-4 (IL-4) is a pleiotropic cytokine which plays a pivotal role in shaping immune responses. IL-4 mediates important proinflammatory functions in asthma, including the IgE isotype switch. IL-4 exerts its biological effects through binding to its receptor (IL-4R) complex, with the alpha chain as the high affinity binding subunit. Soluble IL-4R lacks the transmemberane and cytoplasmic domains, so it cannot induce cellular activation. By acting as a decoy to circulating IL-4 and neutralize its activity, its high specificity and affinity make it ideal as an IL-4 antagonist. Some companies have embarked the clinical research for asthma treatment with the sIL-4R and the result revealed well therapeutic effect. With RNA extracted from human monocyte as the template, The sIL-4R cDNA encoding the extracellular domain of IL-4R a chain was obtained by RT-PCR. Compared with the sIL-4R encoding sequence in GenBank, the nucleotide sequencing analysis indicated that there was a A-->G mutation at 148bp and the mutation caused Ile-->Val at 50th amino acid. According to the references, numerous polymorphisms have been identified in the IL-4R gene and the Ile50Val was the only known extracellular variant of human IL-4R. Then the recombinant vector pPIC9K/sIL-4R was constructed, linearized and introduced into Pichia pastoris GS115 by electroporation. The recombinant sIL-4R was identified by SDS-PAGE, Western blot and Ligand binding blot. The SDS-PAGE and Western blot analysis showed that the apparent molecular weight of expressed sIL-4R was about 30kD. And the Ligand binding blot analysis indicated the expressed sIL-4R had the biological activity. The sIL-4R, which had the biological activity, was successfully secretorily expressed in the Pichia pastoris (GS 115).
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 04/2004; 20(2):197-202.
-
[show abstract]
[hide abstract]
ABSTRACT: Recently in our laboratory, Streptomyces sp. 139 has been identified to produce a new exopolysaccharide designated EPS 139A that shows anti-rheumatic arthritis activity. The strategy of studying EPS 139A biosynthesis is to clone the key gene in the EPS biosynthesis pathway, i.e. the priming glycosyltransferase gene catalyzing the first step of nucleotide sugar transfer. Degenerate primers-based PCR approach was adopted to isolate the putative priming glycosyltransferase gene in Streptomyces sp. 139. According to the genes encoding the priming glycosyltransferases that have been identified in several microorganisms, a multiple alignment of the amino acid sequences of these genes was used to identify regions conserved between all genes. To clone the priming glycosyltransferase gene in Streptomyces sp. 139, degenerate primers were designed from these conserved regions taking into account information on Streptomyces codon usage to amplify an internal DNA fragment of this gene. A distinctive PCR product with the expected size of 0.3 kb was amplified from Streptomyces sp. 139 total genomic DNA. Sequence analysis showed that it is part of a putative priming glycosyltransferase gene and contains the predicted conserved domain B. To isolate the complete priming glycosyltransferase gene, a Streptomyces sp. 139 genomic library was constructed in the E. coli--Streptomyces shuttle vector pOJ446. Using the 0.3 kb PCR product of priming glycosyltransferase gene as a probe, 17 positive colonies were isolated by colony hybridization. A 4.0 kb BamHI fragment from all positive cosmids that hybridized to this probe was sequenced, which revealed the complete priming glycosyltransferase gene. The priming glycosyltransferase gene ste5 (GenBank under accession number AY131229) most likely begins with GTG, preceded by a probable ribosome binding site (RBS), GGGGA. It encodes a 492-amino-acid protein with molecular weight of 54 kDa and isoelectric point of 10.6. The G + C content of ste5 is 73%, close to the average of G + C content (74%) for Streptomyces. Moreover, the preference usage of G or C as third base of codons are found in the ste5, which is in accordance with the Streptomyces codon usage. A BlastP search showed that the C-terminal region of Ste5 shows highly homology with a number of priming glycosyltransferases from many different organisms. Ste5 contains two putative catalytic residues, Glu and Asp (residues 423 and 474) with a spacing of approximately 50 amino acids that conserved in various beta-glycosyltransferases. Moreover, the C-terminal one third of Ste5 contains three domains, A, B and C that is reported to be common to glycosyltransferases. By hydrophilicity plot prediction, the N-terminal two thirds of Ste5 exhibits 5 putative transmembrane domains. To investigate the involvement of the identified polysaccharide gene cluster in EPS 139A biosynthesis, the gene ste5 encoding priming glycosyltransferase was insertionally disrupted by a single-crossover homologous recombination event. A 0.85 kb internal fragment of ste5 was cloned into vector pKC1139 to yield pLY5015 that was transduced into Streptomyces sp. 139. Correct integration in Streptomyces LY1001 ste5- mutant strain was confirmed by Southern hybridization. After fermentation, no EPS 139A could be detected in the cultures of ste5- mutant strain Streptomyces LY1001. Therefore, the gene ste5 identified in this work is involved in the synthesis of the Streptomyces sp. 139 EPS.
Acta Genetica Sinica 09/2003; 30(8):723-9.
-
[show abstract]
[hide abstract]
ABSTRACT: Salmon calcitonin (sCT) is one of the many bioactive peptides that require C-terminal amidation for full biologic activity. To produce fully bioactive sCT in large scale, we constructed Streptomyces lividans [pMSA], an engineering Streptomyces strain. In the expression vector, glycine-extended sCT, the substrate for amidation, and rat alpha-amidating enzyme cDNA were cloned under the control of the strong constitutive promoter from the Streptomyces fradiae aph gene in pIJ680. Both were expressed in a secretory manner by the recombinant strain using the expression and secretion signals of melC1. Extracellularly expressed recombinant sCT was purified to near homogeneity and characterized by enzyme immunoassay, followed by direct amino-terminal sequencing. High-performance liquid chromatography, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, and bioassay in vivo demonstrated purified product to be equivalent to synthetic standard. Thus, the engineered Streptomyces strain can produce bioactive, C-terminal amidated recombinant sCT in the culture supernatant directly. The ease of the recombinant process, as well as its potential for scale-up, makes it adaptable to production demands for sCT, and it may be applied to other bioactive peptides that need C-terminal amidation.
Applied Biochemistry and Biotechnology 09/2003; 110(2):113-23. · 1.94 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Streptomyces sp. 139 produces a new exopolysaccharide (EPS) that shows anti-rheumatic arthritis activity in vivo. To investigate the gene cluster involved in EPS biosynthesis, degenerate primers were designed to amplify an internal fragment of the priming glycosyltransferase gene that catalyzes the first step in EPS biosynthesis. Using this PCR product as probe, positive cosmid clones were selected from a genomic library of Streptomyces sp. 139, which led to the localization of ste (Streptomyces eps) gene cluster on the approximately 65-kb chromosomal region. A 4.0-kb Bam HI fragment from all positive cosmids that hybridized to this probe was sequenced, which revealed two genes encoding the priming glycosyltransferase and UDP-glucose dehydrogenase. The putative priming glycosyltransferase is suggested to catalyze the first step in the biosynthesis of EPS repeating unit and UDP-glucose dehydrogenase is suggested to convert UDP-glucose into UDP-glucuronic acid involved in nucleotide sugar precursor synthesis of EPS biosynthesis.
DNA Sequence 05/2003; 14(2):141-5. · 0.75 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The production of human tumor necrosis factor beta (hTNF beta) in Streptomyces lividans was studied. The expression of hTNF beta uses the transcription, translation and secretion signals of subtilisin inhibitor VSI which is naturally produced by Streptomyces venezuelae CBS762.70. In direct secretory expression cassette, hTNF beta cDNA was fused 2 amino acids after the signal peptidase cleavage site. In fusion expression cassette, hTNF beta cDNA was fused after the total vsi gene. In intracellular expression cassette, hTNF beta cDNA was fused after initiation codon ATG. The expression cassettes were subcloned into Streptomyces multi-copy plasmid pIJ486 respectively and transformed into S. lividans TK24. The recombinant strains were designated as S. lividans (pIJ486-hTNF beta), S. lividans (pIJ486-vsi-hTNF beta) and S. lividans (pIVPA-hTNF beta). The analysis of expressed proteins by Western blotting and biological activity measurements revealed hTNF beta was expressed by the recombinant strains with bioactivity. The molecular weight of directly secreted product is around 16 kDa, and the expression level at 48 hours in NB medium was 0.7 mg/L. Intact product could be obtained when hTNF beta was expressed intracellularly, although it may be degraded to lower molecular weight product when cultivation was prolonged. The intracellular expression level at 48 hours in NB medium was 25.1 mg/L.
Acta Genetica Sinica 04/2003; 30(3):209-14.
-
[show abstract]
[hide abstract]
ABSTRACT: We report the identification and characterization of the ste (Streptomyces eps) gene cluster of Streptomyces sp. 139 required for exopolysaccharide (EPS) biosynthesis. This report is the first genetic work on polysaccharide production in Streptomyces. To investigate the gene cluster involved in exopolysaccharide 139A biosynthesis, degenerate primers were designed to polymerase chain reaction amplify an internal fragment of the priming glycosyltransferase gene that catalyzes the first step in exopolysaccharide biosynthesis. Screening of a genomic library of Streptomyces sp. 139 with this polymerase chain reaction product as probe allowed the isolation of a ste gene cluster containing 22 open reading frames similar to polysaccharide biosynthesis genes of other bacterial species. Involvement of the ste gene cluster in exopolysaccharide biosynthesis was confirmed by disrupting the priming glycosyltransferase gene in Streptomyces sp. 139 to generate non-exopolysaccharide-producing mutants.
FEMS Microbiology Letters 04/2003; 220(1):21-7. · 2.04 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: HDL receptor plays a very important role in reverse cholesterol transport, and it represents a novel therapeutic target for atherosclerosis. For construction of an in vitro high-throughput drug screening model based on competitive receptor--ligand binding, the extracellular domain of HDL receptor was expressed in the methylotropic yeast Pichia pastoris. The DNA fragment encoding for extracellular domain of HDL receptor was cloned by RT-PCR from Hepatoma Bel-7402 total RNA and the nucleotide sequence of the cloned cDNA was verified by inserting it into pGEM-T vector. Then the cDNA was subcloned into pPIC9K, the integrative secretory expression plasmid of Pichia pastoris was constructed, with the methanol-inducible alcohol oxidase promoter and alpha-signal peptide. The recombinant plasmid was transformed into Pichia pastoris GS115 (His- strain) by electroporation. PCR was used to confirm the insertion of HDLR gene into the genome of Mut+ transformants. During cultivation the recombinant strain was induced by 1% methanol and supernatant was analyzed by SDS-PAGE and Western blot. The result showed a specific protein band at approximately 64.5 kDa after 5 days of induction. Then the ligand binding activity was confirmed using the DiI-AcLDL as ligand.
Acta Genetica Sinica 02/2003; 30(1):20-4.
-
[show abstract]
[hide abstract]
ABSTRACT: Using RNA extracted from human umbilical vein endothelium cell as a template, the gene VEGF receptor Flt-1 was amplified by RT-PCR. Recombinant plasmid pSGLgpp-F was constructed and was transformed into S. lividans TK24. With the detection of SDS-PAGE and Western blot, a specific band being same to the reports near 63.6 kd was found. The results showed sFlt-1 was successfully expressed in S. lividans. The result of the binding assay of receptor-ligand for sFlt-1 showed sFlt-1 has the biological activity of binding with its ligand VEGF.
ACTA MICROBIOLOGICA SINICA 09/2002; 42(4):411-7.
-
[show abstract]
[hide abstract]
ABSTRACT: Gene cloning and expression of CTLA-4 was performed in S. lividans with two new types of different signal peptides--vsi and gpp to investigate the secretory expression efficiency of CTLA-4. The hsCTLA-4 gene was fused to the vsi sequences and then inserted into the shuttle vector-pUWL-219. At the same time, the hsCTLA-4 was inserted into the downstream of gpp signal peptide in the plasmid pLNSP. Then the recombinant plasmids were transformed into S. lividans TK24 respectively. The two engineering strains were named as S. lividans [pUWL219-VC] and S. lividans [pLNSP/CTLA-4]. The result of SDS-PAGE and Western blotting show that the recombinant strain S. lividans [pUWL219-VC] and S. lividans [pLNSP/CTLA-4] can express CTLA-4 with about MW 13,000 which is similar to those reported earlier and have immunoactivity. It is the first time that CTLA-4 expression in S. lividans is reported.
Acta Genetica Sinica 02/2002; 29(1):79-83.
-
[show abstract]
[hide abstract]
ABSTRACT: To obtain the expression of human low density lipoprotein receptor ligand binding domain in methylotropic yeast, firstly the DNA fragment encoding for human low density lipoprotein receptor ligand binding domain was amplified by RT-PCR with human hepatoma Bel-7402 total RNA as template. The nucleotide sequencing analysis indicated that the sequence of the cloned DNA fragment was as same as the reported human LDLR cDNA sequence. Then the expression vector pPIC9K-sLDLr was constructed, linearized and introduced into Pichia pastoris GS115 by electroporation. The recombinant sLDLR was identified by SDS-PAGE, Western blot and Ligand binding blot in supernatant of GS115/pPIC9K-sLDLr. The SDS-PAGE and Western blot analysis showed that the apparent molecular weight of expressed sLDLR was about 36 kD. And the ligand binding blot analysis indicated the expressed sLDLR has the biological activity. The sLDLR, which had the biological activity, was successfully secretorily expressed in the Pichia pastoris (GS115).
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 02/2002; 18(1):40-4.
-
[show abstract]
[hide abstract]
ABSTRACT: Ebosin produced by Streptomyces sp. 139 is a novel exopolysaccharide (EPS) with medicinal activity. This paper describes the functional study of ste10, a putative Ebosin biosynthesis gene. ste10 was cloned and expressed in Escherichia coli BL21 and the purified recombinant protein characterized. Ste10 was shown to be able of catalyzing the transfer of amide nitrogen of glutamine to the side chain of aspartate to produce asparagine. Its Km, optimum temperature and pH were determined to be 0.9 mM, 37 °C and 7.38, respectively. After ste10 gene knock-out, the monosaccharide composition of EPS-m produced by the mutant Streptomyces sp. 139 (ste10−) was found changed in comparison with that of Ebosin while its antagonist activity for IL-1R decreased significantly. Based on these results, it is concluded that ste10 codes for an asparagine synthetase which may function as a modificator gene of Ebosin during its biosynthesis.
Enzyme and Microbial Technology.
