Cysteine mediated multimerization of a recombinant dengue E fragment fused to the P64k protein following immobilized metal ion affinity chromatography.
A fragment of the Envelope protein of Dengue 2 virus encoding the amino acid (aa) 286-426 was fused to the P64k protein from Neisseria meningitidis. The hybrid gene (PD5) was expressed in Escherichia coli. The influence of using immobilized metal ion affinity chromatography (IMAC) in the purification process of PD5 was examined. After the elution, PD5 protein formed aggregates of high molecular weight depending on disulfide-bonds. The study of different conditions affecting the redox potential in the system revealed the influence of the copper ions on the multimerization of the protein, whereas metals with minor redox potential-for instance, zinc or nickel ions-did not cause this effect. It was also demonstrated that cysteines involved in this process belonged to the P64k protein. Finally, a PD5 purification process was established reaching a 85% of purity using the Zn(2+) as metal ion in the IMAC. This is a very useful finding due to the wide use of P64k as a carrier protein and the advantages of the IMAC as a chromatographic process.
Centro de Ingeniería Genética y Biotecnología, División de Vacunas, Havana, Cuba. c.lopez@cigb.edu.cu
Carlos
C
López
Jorge
J
Sánchez
Lisset
L
Hermida
Aída
A
Zulueta
Gabriel
G
Márquez
D002614:Chelating Agents / Q000737:chemistry; D002846:Chromatography, Affinity; D003300:Copper / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D003716:Dengue Virus / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D020450:Heavy Ions; D009345:Neisseria meningitidis / Q000737:chemistry*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014759:Viral Envelope Proteins / Q000302:isolation & purification*; D015032:Zinc / Q000737:chemistry
D002614:Chelating Agents; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D003300:Copper; D015032:Zinc; D003545:Cysteine