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
VL domains. Here we apply rational
stability engineering by consensus sequence analysis to
VH domains. Isolated
VH domains tend to aggregate
more easily than VL 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 VH
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 VH
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 VL domain. This
success provides further evidence that consensus sequence
engineering is a rational, plannable route to the construction
of intrabodies.