Purification and conformational properties of a human interferon alpha2b produced in Escherichia coli.
Recombinant human interferon alpha2b was expressed intracellularly in Escherichia coli as insoluble aggregates using a new expression vector, and was purified to homogeneity using essentially two-step chromatographic procedures, i.e. immobilized metal-ion-affinity chromatography and reversed-phase HPLC. The established purification process is highly reproducible and leads to a total recovery of approx. 12% with a specific biological activity of higher than 1x10(8) i.u./mg of protein, which is comparable with the international requirement for interferon alpha2b. For purified protein we report conformational stability as a function of pH and temperature using differential scanning calorimetry and CD. Thermal unfolding as a function of pH showed only one endotherm at a temperature higher than 45 degrees C, and was reversible at pH 2-3.75 and irreversible at pH 4-10. At pH 7.0, the most stable condition, the conformational stability depends on protein concentration and ionic strength. The highly helical secondary structure is very conserved over the whole pH range studied, including at high temperatures.
Beldarraín
A
A
Processes and Systems Evaluation Department, Production Plant, CIGB (Center for Genetic Engineering and Biotechnology), P.O. Box 6162, Havana, Cuba. alejandro.beldarrain@cigb.edu.cu
Cruz
Y
Y
Cruz
O
O
Navarro
M
M
Gil
M
M
D002152:Calorimetry, Differential Scanning / Q000379:methods; D002847:Chromatography, Agarose / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D009994:Osmolar Concentration; D011487:Protein Conformation; D015202:Protein Engineering / Q000592:standards; D011994:Recombinant Proteins; D015203:Reproducibility of Results
D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins