Folding and stability of chimeric immunofusion VL-barstar

Y. I. Tsybovsky, D. V. Shubenok, O. A. Stremovskiy, S. M. Deyev, S. P. Martsev

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)939-948
Number of pages10
JournalBiochemistry (Moscow)
Volume69
Issue number9
DOIs
Publication statusPublished - Sep 2004
Externally publishedYes

Fingerprint

Proteins
Conformations
Bispecific Antibodies
Fluorescence Spectrometry
Inclusion Bodies
Protein Folding
Differential Scanning Calorimetry
Ferritins
Ribonucleases
Recombinant Proteins
Escherichia coli
Purification
Bacillus amyloliquefaciens barstar protein
Molten materials
Immunoglobulins
Differential scanning calorimetry
Fusion reactions
Fluorescence
Monoclonal Antibodies
Association reactions

Keywords

  • Antiferritin antibody
  • Barstar
  • Chimeric immunofusions
  • Differential scanning calorimetry
  • Protein folding
  • Recombinant antibody
  • VL domain

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry

Cite this

Tsybovsky, Y. I., Shubenok, D. V., Stremovskiy, O. A., Deyev, S. M., & Martsev, S. P. (2004). Folding and stability of chimeric immunofusion VL-barstar. Biochemistry (Moscow), 69(9), 939-948. https://doi.org/10.1023/B:BIRY.0000043536.94292.7d

Folding and stability of chimeric immunofusion VL-barstar. / Tsybovsky, Y. I.; Shubenok, D. V.; Stremovskiy, O. A.; Deyev, S. M.; Martsev, S. P.

In: Biochemistry (Moscow), Vol. 69, No. 9, 09.2004, p. 939-948.

Research output: Contribution to journalArticle

Tsybovsky, YI, Shubenok, DV, Stremovskiy, OA, Deyev, SM & Martsev, SP 2004, 'Folding and stability of chimeric immunofusion VL-barstar', Biochemistry (Moscow), vol. 69, no. 9, pp. 939-948. https://doi.org/10.1023/B:BIRY.0000043536.94292.7d
Tsybovsky, Y. I. ; Shubenok, D. V. ; Stremovskiy, O. A. ; Deyev, S. M. ; Martsev, S. P. / Folding and stability of chimeric immunofusion VL-barstar. In: Biochemistry (Moscow). 2004 ; Vol. 69, No. 9. pp. 939-948.
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