Engineering an ultra-stable affinity reagent based on Top7

Abstract

Antibodies are widely used for diagnostic and therapeutic applications because of their sensitive and specific recognition of a wide range of targets; however, their application is limited by their structural complexity. More demanding applications require greater stability than can be achieved by immunoglobulin-based reagents. Highly stable, protein-based affinity reagents are being investigated for this role with the goal of identifying a suitable scaffold that can attain specificity and sensitivity similar to that of antibodies while performing under conditions where antibodies fail. We have engineered Top7--a highly stable, computationally designed protein--to specifically bind human CD4 by inserting a peptide sequence derived from a CD4-specific antibody. Molecular dynamics simulations were used to evaluate the structural effect of the peptide insertion at a specific site within Top7 and suggest that this Top7 variant retains conformational stability over 100 degrees C. This engineered protein specifically binds CD4 and, consistent with simulations, is extremely resistant to thermal and chemical denaturation--retaining its secondary structure up to at least 95 degrees C and requiring 6 M guanidine to completely unfold. This CD4-specific protein demonstrates the functionality of Top7 as a viable scaffold for use as a general affinity reagent which could serve as a robust and inexpensive alternative to antibodies.

Molecular basis of the structural stability of a Top7-based scaffold at extreme pH and temperature conditions

“…Chemical and thermal unfolding measurements showed that the engineered protein maintains stability at neutral pH, with no significant change in the free energy of unfolding when compared to native Top7. Of most relevance, ELISA experiments revealed that the engineered Top7 binds to CD4 whereas the native protein does not [11]. These findings demonstrated that Top7 displays considerable potential as an affinity reagent scaffold where binding sites for targets of interest can be grafted.…”

“…The Top7 K39E K40E K55E (referred to here as Top7) and Top7 CB1 proteins were expressed and purified as previously described [11]. Proteins were buffer exchanged (HiTrap Desalting column, GE Healthcare) into phosphate buffered saline (PBS, 100 mM NaH 2 PO 4 and150 mM NaCl) at pH 7.0 or pH 2.0.…”

“…This limitation has precluded the development of protein-based affinity reagents that can tolerate environmental extremes and extend the utility of these agents to applications outside of the highly controlled laboratory setting [53]. An alternative approach is the design of non-natural protein scaffolds stable under adverse environments [11]. A promising candidate for such scaffolds is the protein Top7, a de novo designed protein with a novel sequence and fold ( Fig.…”

“…It has been further shown that the three-dimensional structures of nonhomologous amino acid sequences designed to adopt the Top7 fold exhibit high thermodynamic stability [19,77]. We have previously engineered a highly stable variant of Top7 capable to recognize the glycoprotein CD4 [11]. The novel functionality has been implemented by the insertion of an 8-residue sequence derived from the anti-CD4 13B8.2 paratope [2].…”

“…In the present study, a combined experimental-computational approach was used to examine the effect of low pH, high temperature and concerted mutations on the stability and structural dynamics of the Top7-based scaffold [11]. Far-UV CD spectroscopy was used to evaluate the chemo and thermostability of the Top7 scaffold.…”