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.
Submitter: Shu-Ching Ou
Submission Date: Oct. 23, 2018, 2:55 p.m.
|Number of data points||24|
|Proteins||Immunoglobulin heavy chain domain|
|Assays/Quantities/Protocols||Experimental Assay: t50: time after which 50% of the protein has become insoluble ; Experimental Assay: Urea50: concentration of denaturant at which 50% of the folded domain fluorescence signal is lost ; Experimental Assay: T50: temperature at which 50% of the protein has become insoluble ; Derived Quantity: SD of T50: temperature at which 50% of the protein has become insoluble|
|Libraries||Fifty percent survival of successively stabilized VH domains|