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.

Submission Details

ID: EcHNySVP3

Submitter: Shu-Ching Ou

Submission Date: Oct. 23, 2018, 2:55 p.m.

Version: 1

Publication Details
Wirtz P;Steipe B,Protein Sci (1999) Intrabody construction and expression III: engineering hyperstable V(H) domains. PMID:10595527
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)