Abstract：
: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.
journal_name
Mol Biotechnoljournal_title
Molecular biotechnologyauthors
Newton DL,Rybak SMdoi
10.1385/MB:20:1:063keywords:
subject
Has Abstractpub_date
2002-01-01 00:00:00pages
63-76issue
1eissn
1073-6085issn
1559-0305pii
MB:20:1:063journal_volume
20pub_type
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