Intrabody construction and expression III: Engineering hyperstable V_H domains

Abstract

The folding of immunoglobulin domains requires the formation of a conserved structural disulfide. Therefore, as a general rule, they cannot be functionally expressed in the reducing environment of the cellular cytoplasm. We have previously reported that stability engineering can lead to the cytoplasmic expression of functional immunoglobulin V_L domains. Here we apply rational stability engineering by consensus sequence analysis to V_H domains. Isolated V_H domains tend to aggregate more easily than V_L domains; they do not refold quantitatively and are generally more difficult to handle in vitro. To overcome these problems, we successfully predicted and experimentally verified several stabilizing point mutations in the V_H domain of a designed, catalytic Fv fragment. The effect of single mutations was additive, and they could be combined in a prototype domain with significantly improved stability against chemical denaturation and a 20-fold increased half time of irreversible thermal denaturation, at physiological temperature. This stabilized, isolated V_H domain could be expressed solubly in the reducing cellular cytoplasm of Escherichia coli, at a yield of approximately 1.2 mg/L of shake flask culture. It remains fully functional, as evidenced by the successful reconstitution of an esterolytic Fv fragment with the V_L domain. This success provides further evidence that consensus sequence engineering is a rational, plannable route to the construction of intrabodies.