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high-level expression of a soluble snake venom enzyme, gloshedobin, in e. coli in the presence of metal ions.the mature gene of gloshedobin, a snake venom thrombin-like enzyme from the snake, gloydius shedaoensis, was cloned and expressed in strain e. coli bl21 (de3). having been induced by iptg, the recombinant gloshedobin was in both soluble and insoluble forms. to avoid inclusion body formation, expression was optimized at 25 degrees c. furthermore, a 50% increase in solubilization of the target protein was obtained by adding 0.1 mm mg2+ to the medium. the purified recombinant gloshedobin gave a 44 ...200312882153
soluble expression, purification, and characterization of gloydius shedaoensis venom gloshedobin in escherichia coli by using fusion partners.gloshedobin, a thrombin-like enzyme from the venom of gloydius shedaoensis, is usually produced as inclusion bodies in escherichia coli cell. in this work, gloshedobin was separately fused with three fusion partners nusa, gst, and trxa at its n terminus and then was expressed as fusion proteins in e. coli. the results showed that the nusa was the most efficient fusion partner to improve the solubility of recombinant gloshedobin. the purified nusa-fused gloshedobin with an overall yield of 64.6% ...201019639313
chaperone-assisted column refolding of gloshedobin with the use of refolding cocktail.gloshedobin, a recently isolated thrombin-like enzyme from the snake venom of gloydius shedaoensis, is expressed mainly in the form of inclusion bodies (ibs) in escherichia coli due to its cysteine-rich nature. following extraction and solubilization of the ibs, one-step immobilized metal affinity chromatography purification produces highly purified (>99%) denatured-solubilized gloshedobin ready to enter the subsequent refolding process. however, the traditional dilution or column refolding stra ...200819004452
purification, gene cloning and expression of an acidic phospholipase a2 from agkistrodon shedaoensis zhao.a protein with the activity of phospholipase a(2) named asapla(2) was purified to homogeneity from the venom of agkistrodon shedaoensis zhao through deae-sepharose cl-6b anion exchange column, source s, and mono q fplc. its molecular weight was estimated to be 19 kd by sds-page, and its pi was about 3.5 by ief analysis. it inhibited the platelet aggregation that was induced by 1 micromol/ l adp, and the ic(50) was determined to be 6 micromol/l. degenerating primer was designed and synthesized ac ...200414732871
expression of gloshedobin, a thrombin-like enzyme from the venom of gloydius shedaoensis, in escherichia coli.gloshedobin, a thrombin-like enzyme from the venom of gloydius shedaoensis, was expressed in escherichia coli using expression vector pet-32a(+). the gene was expressed under t7 promotor with a fusion partner of thx.tag and a 6xhis.tag at its 5' terminal. after induction by iptg for 6 h, the recombinant enzyme was expressed in the cytoplasm. expression at 25 degrees c gave twice the amount of recombinant gloshedobin in cytoplasm than at 37 degrees c.200312882282
sequence and bioinformatic characterization of expressed sequence tags originated from gloydius shedaoensis shedaoensis venom gland.first, a cdna library from gloydius shedaoensis shedaoensis venom gland (gssg) was constructed using smart technique. the total rna in a pair of gssgs was separated and the mrna was further isolated from it. the first-strand cdna was synthesized through powerscript(™) reverse transcriptase by a cds iii/3' pcr primer. a smart iv(™) -oligonucleotide was used as the template for the synthesis of double-strand cdna and amplified by long-distance polymerase chain reaction (ld-pcr). ld-pcr product was ...201323408674
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