Wang H

Immunoaffinity purification of polyepitope proteins against Plasmodium falciparum with chicken IgY specific to their C-terminal epitope tag

Developing a polyepitope vaccine, a chimeric protein which contains diverse types of antigenic epitopes, is a promising strategy to prevent malaria. Previously, we had constructed a library of polyeptitope chimeric genes against Plasmodium falciparum without any protein tags. In an attempt to develop an efficient and universal procedure for purification of polyepitope chimeric proteins, we assembled an immunoaffinity chromatography (IAC) column with affinity-purified specific polyclonal IgY (mpIgY) antibodies that recognized the same C-terminal epitope tag of chimeric proteins in the library. A single-step and universal protocol was established and successfully applied for the purification of chimeric proteins. Using this protocol, chimeric proteins were specifically purified from an Escherichia coli expression system, and the purity and authenticity were verified by gel electrophoresis and Western blot analysis. Moreover, the comparison between this IAC method and the conventional chromatography, using two anion exchange columns followed by a step of gel filtration, showed that the new method was more efficient with an 8-fold greater yield. The results suggest that this IAC method will be an efficient approach for the purifications of polyepitope vaccine candidates against P. falciparum in our future study, and also be valuable for other similar applications.

Molecular cloning, expression, and purification of SARS-CoV nsp13.

Abstract The SARS-nsp13 protein was identified as an mRNA cap1 methyltransferase. In this study, the nsp13 gene was cloned from the SARS-CoV PUMC02 strain viral RNA by RT-PCR, and inserted into the expression plasmid pET30a(+). The recombinant plasmid pET30a(+)-nsp13 was confirmed by restriction enzymes and sequencing analysis, and transformed into Escherichia coli BL21(DE3). The His-tag-fused protein was expressed by induction of 0.5mM IPTG and purified by a single Ni2+ affinity chromatography. The protein was validated by western blot and MS analysis. A large quantity of the nsp13 protein obtained with this method may be useful for further study of its structure and function.