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.
- Differential scanning calorimetry
- Molten globule
- Protein folding
- VH domain