Folding and Stability of Chimeric Immunofusion VL-Barstar.

Autor: Tsybovsky, Y. I., Shubenok, D. V., Stremovskiy, O. A., Deyev, S. M., Martsev, S. P.
Předmět:
Zdroj: Biochemistry (00062979); Sep2004, Vol. 69 Issue 9, p939-948, 10p
Abstrakt: A chimeric protein VL-barstar that comprises the VL domain of anti-human ferritin monoclonal antibody F11 and barstar, the naturally occurring inhibitor of bacterial RNase barnase, has been constructed for study of structure–function characteristics of chimeric immunoglobulin fused proteins. Such chimeric constructs may be potentially employed for development of bivalent/bispecific antibodies on the basis of the high affinity interaction between barstar and barnase (the association constant is about 1014 M–1). We have developed a protocol for VL-barstar expression in E. coli and purification and refolding from inclusion bodies that yields a homogeneous and soluble form of this protein. Differential scanning calorimetry in combination with fluorescence and CD spectroscopy revealed that the VL-barstar formed well-resolved ordered secondary and compact tertiary structures. However, partial loss of tertiary interactions resulted in low stability of the recombinant protein and the lack of functional activity of the two constituent modules. These conformational features suggest that the protein might be referred to the class of native molten globules, which comprises partially unfolded conformations stabilized under physiological conditions. Since individually expressed VL domain and barstar retain completely folded conformation and stable spatial structure, the incomplete folding of the chimeric protein may be attributed to interaction between heterologous domains, which appears at the folding stage preceding formation of a system of tertiary interactions in both structural modules. The results provide evidence for non-native interactions between heterologous modules that may occur in chimeric proteins composed of taxonomically distinct fusion partners. [ABSTRACT FROM AUTHOR]
Databáze: Complementary Index