Fusion of barnase to antiferritin antibody F11 VH domain results in a partially folded functionally active protein

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

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

A chimeric protein, VH-barnase, was obtained by fusing the VH domain of anti-human ferritin monoclonal antibody F11 to barnase, a bacterial RNase from Bacillus amyloliquefaciens. After refolding from inclusion bodies, the fusion protein formed insoluble aggregates. Off-pathway aggregation was significantly reduced by adding either purified GroEL/GroES chaperones or arginine, with 10-12-fold increase in the yield of the soluble protein. The final protein conformation was identical by calorimetric criteria and CD and fluorescence spectroscopy to that obtained without additives, thus suggesting that VH-barnase structure does not depend on folding conditions. Folding of VH-barnase resulted in a single calorimetrically revealed folding unit, the so-called "calorimetric domain", with conformation consistent with a molten globule that possessed well-defined secondary structure and compact tertiary conformation with partial exposure of hydrophobic patches and low thermodynamic stability. The unique feature of VH-barnase is that, despite the partially unfolded conformation and coupling into a single "calorimetric domain", this immunofusion retained both the antigen-binding and RNase activities that belong to the two heterologous domains. © 2009 Pleiades Publishing, Ltd.
Original languageEnglish
Pages (from-to)672-680
Number of pages9
JournalBiochemistry (Moscow)
Volume74
Issue number6
DOIs
Publication statusPublished - 1 Jun 2009

Fingerprint

Fusion reactions
Conformations
Antibodies
Proteins
Protein Conformation
Fluorescence Spectrometry
Inclusion Bodies
Fluorescence spectroscopy
Ferritins
Ribonucleases
Thermodynamics
Arginine
Bacillus amyloliquefaciens ribonuclease
Molten materials
Thermodynamic stability
Agglomeration
Monoclonal Antibodies
Antigens

Keywords

  • Barnase
  • Differential scanning calorimetry
  • Immunofusion
  • Molten globule
  • Protein folding
  • VH domain

Cite this

Fusion of barnase to antiferritin antibody F11 VH domain results in a partially folded functionally active protein. / Shubenok, D. V.; Tsybovsky, Y. I.; Stremovskiy, O. A.; Deyev, S. M.; Martsev, S. P.

In: Biochemistry (Moscow), Vol. 74, No. 6, 01.06.2009, p. 672-680.

Research output: Contribution to journalArticle

Shubenok, D. V. ; Tsybovsky, Y. I. ; Stremovskiy, O. A. ; Deyev, S. M. ; Martsev, S. P. / Fusion of barnase to antiferritin antibody F11 VH domain results in a partially folded functionally active protein. In: Biochemistry (Moscow). 2009 ; Vol. 74, No. 6. pp. 672-680.
@article{fe337615e25f4e6790a35deb9b567a52,
title = "Fusion of barnase to antiferritin antibody F11 VH domain results in a partially folded functionally active protein",
abstract = "A chimeric protein, VH-barnase, was obtained by fusing the VH domain of anti-human ferritin monoclonal antibody F11 to barnase, a bacterial RNase from Bacillus amyloliquefaciens. After refolding from inclusion bodies, the fusion protein formed insoluble aggregates. Off-pathway aggregation was significantly reduced by adding either purified GroEL/GroES chaperones or arginine, with 10-12-fold increase in the yield of the soluble protein. The final protein conformation was identical by calorimetric criteria and CD and fluorescence spectroscopy to that obtained without additives, thus suggesting that VH-barnase structure does not depend on folding conditions. Folding of VH-barnase resulted in a single calorimetrically revealed folding unit, the so-called {"}calorimetric domain{"}, with conformation consistent with a molten globule that possessed well-defined secondary structure and compact tertiary conformation with partial exposure of hydrophobic patches and low thermodynamic stability. The unique feature of VH-barnase is that, despite the partially unfolded conformation and coupling into a single {"}calorimetric domain{"}, this immunofusion retained both the antigen-binding and RNase activities that belong to the two heterologous domains. {\circledC} 2009 Pleiades Publishing, Ltd.",
keywords = "Barnase, Differential scanning calorimetry, Immunofusion, Molten globule, Protein folding, VH domain",
author = "Shubenok, {D. V.} and Tsybovsky, {Y. I.} and Stremovskiy, {O. A.} and Deyev, {S. M.} and Martsev, {S. P.}",
year = "2009",
month = "6",
day = "1",
doi = "10.1134/S0006297909060121",
language = "English",
volume = "74",
pages = "672--680",
journal = "Biochemistry. Biokhimiia",
issn = "0006-2979",
publisher = "Maik Nauka-Interperiodica Publishing",
number = "6",

}

TY - JOUR

T1 - Fusion of barnase to antiferritin antibody F11 VH domain results in a partially folded functionally active protein

AU - Shubenok, D. V.

AU - Tsybovsky, Y. I.

AU - Stremovskiy, O. A.

AU - Deyev, S. M.

AU - Martsev, S. P.

PY - 2009/6/1

Y1 - 2009/6/1

N2 - A chimeric protein, VH-barnase, was obtained by fusing the VH domain of anti-human ferritin monoclonal antibody F11 to barnase, a bacterial RNase from Bacillus amyloliquefaciens. After refolding from inclusion bodies, the fusion protein formed insoluble aggregates. Off-pathway aggregation was significantly reduced by adding either purified GroEL/GroES chaperones or arginine, with 10-12-fold increase in the yield of the soluble protein. The final protein conformation was identical by calorimetric criteria and CD and fluorescence spectroscopy to that obtained without additives, thus suggesting that VH-barnase structure does not depend on folding conditions. Folding of VH-barnase resulted in a single calorimetrically revealed folding unit, the so-called "calorimetric domain", with conformation consistent with a molten globule that possessed well-defined secondary structure and compact tertiary conformation with partial exposure of hydrophobic patches and low thermodynamic stability. The unique feature of VH-barnase is that, despite the partially unfolded conformation and coupling into a single "calorimetric domain", this immunofusion retained both the antigen-binding and RNase activities that belong to the two heterologous domains. © 2009 Pleiades Publishing, Ltd.

AB - A chimeric protein, VH-barnase, was obtained by fusing the VH domain of anti-human ferritin monoclonal antibody F11 to barnase, a bacterial RNase from Bacillus amyloliquefaciens. After refolding from inclusion bodies, the fusion protein formed insoluble aggregates. Off-pathway aggregation was significantly reduced by adding either purified GroEL/GroES chaperones or arginine, with 10-12-fold increase in the yield of the soluble protein. The final protein conformation was identical by calorimetric criteria and CD and fluorescence spectroscopy to that obtained without additives, thus suggesting that VH-barnase structure does not depend on folding conditions. Folding of VH-barnase resulted in a single calorimetrically revealed folding unit, the so-called "calorimetric domain", with conformation consistent with a molten globule that possessed well-defined secondary structure and compact tertiary conformation with partial exposure of hydrophobic patches and low thermodynamic stability. The unique feature of VH-barnase is that, despite the partially unfolded conformation and coupling into a single "calorimetric domain", this immunofusion retained both the antigen-binding and RNase activities that belong to the two heterologous domains. © 2009 Pleiades Publishing, Ltd.

KW - Barnase

KW - Differential scanning calorimetry

KW - Immunofusion

KW - Molten globule

KW - Protein folding

KW - VH domain

UR - http://www.scopus.com/inward/record.url?scp=67650081362&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=67650081362&partnerID=8YFLogxK

U2 - 10.1134/S0006297909060121

DO - 10.1134/S0006297909060121

M3 - Article

C2 - 19645673

AN - SCOPUS:67650081362

VL - 74

SP - 672

EP - 680

JO - Biochemistry. Biokhimiia

JF - Biochemistry. Biokhimiia

SN - 0006-2979

IS - 6

ER -