Articles
ISOLATION OF ATTACIN E PROTEIN FROM AN ESCHERICHIA COLI EXPRESSION VECTOR AND PRODUCTION OF ITS ANTIBODY FOR USE IN IMMUNOASSAYS
To address this problem, we attempted to produce attacin E in the pRSET Escherichia coli expression vector (Figure 1). Due to stringent control of gene transcription, this vector allows for expression of proteins that are normally toxic to bacterial cells.
High level expression from the T7 promoter results from injection with an M13 phage carrying the T7 polymerase gene.
In addition, pRSET is designed to produce recombinant proteins fused to a short N-terminal peptide containing six histidine residues in tandem, to allow for purification from crude cell lysate by affinity chromatography; and an internal enterokinase cleavage site, to allow for removal of the N-terminal peptide from the fusion protein after purification.
The attacin E gene was cloned into the pRSETB bacterial expression vector to yield pRSETBAtt.
After infection with M13/T7 phage, E.coli JM109 (pRSETBAtt) produced a fusion attacin protein of the predicted size that reacted both to attacin and N-terminal peptide specific antibodies.
In contrast to previously published reports that attacin expressed in E. coli adversely affected cell growth, expression of fusion attacin did not adversely affect growth.
An antibacterial activity assay indicated that fusion attacin (0.2 to 3 μg) was inactive, although attacins in hemolymph from H. cecropia were active.
Attacin E could not be isolated from fusion attacin due to degradation of attacin during enterokinase digestion.
However, polyclonal antisera to the attacin fusion protein had specific immunointeraction against attacins in hemolymph and those expressed in transgenic apple lines.
Attacin expression levels in transgenic lines were estimated using densitometric analysis after immunoblotting.