Abstract
| Original language | English |
|---|---|
| Pages (from-to) | 672-680 |
| Number of pages | 9 |
| Journal | Biochemistry (Moscow) |
| Volume | 74 |
| Issue number | 6 |
| DOIs | |
| Publication status | Published - 1 Jun 2009 |
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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 journal › Article
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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
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U2 - 10.1134/S0006297909060121
DO - 10.1134/S0006297909060121
M3 - Article
VL - 74
SP - 672
EP - 680
JO - Biochemistry. Biokhimiia
JF - Biochemistry. Biokhimiia
SN - 0006-2979
IS - 6
ER -