Purification of fusion proteins expressed by pEX3 and a truncated pEX3 derivative

Abstract

A derivative of the pEX3 expression vector was constructed that codes for the first 407 amino acids of the 1051 amino acids of the pEX3 fusion protein. The amount of truncated fusion protein (40 mg/g cells), obtained by expression in E. coli, was similar to that produced by the original pEX3 vector. The truncated fusion protein was purified more easily from E. co/i contaminants than the original fusion protein by washing with 2 M urea and 0.5% Triton X-100.pEX fusion protein; Truncated form; Protein purification

Antigenic analysis of the coat protein of beet necrotic yellow vein virus by means of monoclonal antibodies

“…Purified virus preparations (approx 2 mg/ml) were treated with 0.2% trypsin in 200 mM-Tris HC1 pH 6-8 for 150 min at 37 °C or with 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.001% bromophenol blue, 0.125 M-Tris HCI pH 8-8 (Laemmli & Favre, 1973) for 3 rnin at 100°C. Fusion proteins containing either aa 1 to 103 or 104 to 188 of the BNYVV coat protein at the C terminus of a truncated cro-lacl lacZ sequence were obtained by cloning the corresponding cDNA sequences of the viral coat protein gene into the truncated derivative of the pEX3 vector described by Kocken et al (1988). The fusion proteins were expressed in, and purified from, E. coli strain POP 2136 following essentially the procedure of Kocken et al (1988).…”

“…Fusion proteins containing either aa 1 to 103 or 104 to 188 of the BNYVV coat protein at the C terminus of a truncated cro-lacl lacZ sequence were obtained by cloning the corresponding cDNA sequences of the viral coat protein gene into the truncated derivative of the pEX3 vector described by Kocken et al (1988). The fusion proteins were expressed in, and purified from, E. coli strain POP 2136 following essentially the procedure of Kocken et al (1988). The pellets obtained after washing the inclusion bodies in 2M-urea, 1 mM-EDTA, 100mM-NaC1, 50 mM-Tris HCI pH 8.0, were resuspended in 9 volumes of 6 M-urea, 0-5~ (v/v) Triton X-100, 10mM-EDTA, 100mM-NaCI, 50mM-Tris-HC1 pH8.0 and centrifuged at 12000g for 20min at 4°C.…”

Immunodetection of the proteins encoded by grapevine chrome mosaic nepovirus RNA2

“…Induction of expression and purification of fusion proteins (in the form of insoluble cellular inclusions) were performed essentially as described by Kocken et al (1988). The final pellet was resuspended in gel loading buffer (60 mMTris-HC1 pH 6-8, 4% SDS, 10~ 2-mercaptoethanol, 15~ glycerol, bromophenol blue) and the fusion protein was further purified by preparative denaturing PAGE (Laemmli, 1970).…”

Epitope mapping on fragments of beet necrotic yellow vein virus coat protein

“…A cDNA clone of the BNYVV CP gene (pCG700) (Ehlers et aL, 1991) served as the starting material for the construction of deletion clones using the modified expression vector pEX2 (Kocken et al, 1988) (Table 1). The deletion clones were obtained by restriction endonuclease subfragment excision, using the natural sites available in the cDNA of the CP gene 0001-0613 © 1992 SGM t pGC700 is described by Ehlers et al (1991).…”