fulltext.study @t Gmail

Engineering a novel, stable dimeric streptavidin with lower isoelectric point

Paper ID Volume ID Publish Year Pages File Format Full-Text
25375 43571 2007 13 PDF Available
Title
Engineering a novel, stable dimeric streptavidin with lower isoelectric point
Abstract

We have engineered a soluble, stable two-chain dimeric streptavidin (TCD) in Escherchia coli. Examination of the three-dimensional structure of streptavidin aided by empirical binding free-energy calculations helped us to select mutations at subunit interfaces that dissociate the native tetramer and stabilize the desired dimer. We chose positions W120, L124, V125 and H127 and mutated them to 120D/124D/125D/127D (TCD-1); 120D/124N/125S/127D (TCD-2); and 120D/124D/125S/127D (TCD-3). The H127D mutation creates electrostatic repulsion that disrupts the dimer–dimer interface, but leaves it very hydrophobic. Therefore, W120, L124 and V125 were mutated to hydrophilic residues to increase dimer solubility. Among the three candidates, TCD-2 gave the best result: a stable, active dimer with Kd for biotin of ∼1 × 10−7 M after purification by gel-filtration chromatography. The experimental results confirm the possibility of rational engineering of low-pI dimeric streptavidins. Reduced-size streptavidin mutants with a net negative charge may be more suitable than antibodies or wild-type streptavidin for the targeting step in radioimmunotherapy because they should clear faster from the bloodstream and the kidney.

Keywords
FPLC, fast-protein liquid chromatography; TCD, two-chain dimer; SCD, single-chain dimerBiotin binding; Dimeric streptavidin; Molecular modeling; Protein engineering; Protein–protein interaction; Radioimmunotherapy
First Page Preview
Engineering a novel, stable dimeric streptavidin with lower isoelectric point
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 128, Issue 2, 1 February 2007, Pages 213–225
Authors
, , , , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering