Linked Life Data

11876300

Source:http://linkedlifedata.com/resource/pubmed/id/11876300

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2002-3-5
pubmed:abstractText
This article describes the construction, expression, and purification of RNase single-chain antibody fusion proteins. To construct a fusion protein, the gene for each moiety, the RNase and the binding ligand, is modified separately to contain complementary DNA encoding a 13 amino acid spacer that separates the RNase from the binding moiety. Appropriate restriction enzyme sites for cloning into the vector are also added. The modified DNA is combined and fused using the PCR technique of splicing by overlap extension (1). The resulting DNA construct is expressed in inclusion bodies in BL21(DE3) bacteria that are specifically engineered for the expression of toxic proteins (2). After isolation and purification of the inclusion bodies, the fusion protein is solubilized, denatured, and renatured. The renatured RNase fusion protein mixture is purified to homogeneity by two chromatography steps. The first column, a CM-Sephadex C-50 or a heparin Sepharose column, eliminates the majority of contaminating proteins while the second column, an affinity column (Ni2+-NTA agarose), results in the final purification of the RNase fusion protein.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
1073-6085
pubmed:author
pubmed:issnType
Print
pubmed:volume
20
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
63-76
pubmed:dateRevised
2005-11-16
pubmed:meshHeading
pubmed:year
2002
pubmed:articleTitle
Preparation of recombinant RNase single-chain antibody fusion proteins.
pubmed:affiliation
SAIC Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, MD 21702, USA.
pubmed:publicationType
Journal Article, Review