Linked Life Data

16854593

Source:http://linkedlifedata.com/resource/pubmed/id/16854593

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2006-11-6
pubmed:abstractText
The two-kringle domain of tissue-type plasminogen activator (TK1-2) has been identified as a potent angiogenesis inhibitor by suppressing endothelial cell proliferation, in vivo angiogenesis, and in vivo tumor growth. Escherichia coli-derived, non-glycosylated TK1-2 more potently inhibits in vivo tumor growth, whereas Pichia expression system is more efficient for producing TK1-2 as a soluble form, albeit accompanying N-glycosylation. Therefore, in order to avoid immune reactivity and improve in vivo efficacy, we expressed the non-glycosylated form of TK1-2 in Pichia pastoris and evaluated its activity in vitro. When TK1-2 was mutated at either Asn(117) or Asn(184) by replacing with Gln, the mutated proteins produced the glycosylated form in Pichia, of which sugar moiety could be deleted by endoglycosidase H treatment. When both sites were replaced by Gln, the resulting mutant produced a non-glycosylated protein, NQ-TK1-2. Secreted NQ-TK1-2 was purified from the culture broth by sequential ion exchange chromatography using SP-sepharose, Q-spin, and UNO-S1 column. The purified NQ-TK1-2 migrated as a single protein band of approximately 20 kDa in SDS-PAGE and its mass spectrum showed one major peak of 19,950.71 Da, which is smaller than those of two glycosylated forms of wild type TK1-2. Functionally, the purified NQ-TK1-2 inhibited endothelial cell proliferation and migration stimulated by bFGF and VEGF, respectively. Therefore, the results suggest that non-glycosylated TK1-2 useful for the treatment of cancer can be efficiently produced in Pichia, with retaining its activity.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
1046-5928
pubmed:author
pubmed:issnType
Print
pubmed:volume
50
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1-8
pubmed:meshHeading
pubmed-meshheading:16854593-Angiogenesis Inhibitors, pubmed-meshheading:16854593-Cell Movement, pubmed-meshheading:16854593-Cell Proliferation, pubmed-meshheading:16854593-Cells, Cultured, pubmed-meshheading:16854593-Dose-Response Relationship, Drug, pubmed-meshheading:16854593-Endothelial Cells, pubmed-meshheading:16854593-Fibroblast Growth Factor 2, pubmed-meshheading:16854593-Gene Expression Regulation, Enzymologic, pubmed-meshheading:16854593-Humans, pubmed-meshheading:16854593-Kringles, pubmed-meshheading:16854593-Mutagenesis, Site-Directed, pubmed-meshheading:16854593-Pichia, pubmed-meshheading:16854593-Structure-Activity Relationship, pubmed-meshheading:16854593-Thymidine Kinase, pubmed-meshheading:16854593-Tissue Plasminogen Activator, pubmed-meshheading:16854593-Vascular Endothelial Growth Factors
pubmed:year
2006
pubmed:articleTitle
Expression of the non-glycosylated kringle domain of tissue type plasminogen activator in Pichia and its anti-endothelial cell activity.
pubmed:affiliation
Cancer Research Institute, College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, Non-U.S. Gov't