Intrabody construction and expression. I. The critical role of VL domain stability.


We have constructed a panel of hyperstable immunoglobulin VL domains by a rational approach of consensus sequence engineering and combining stabilizing point mutations. These prototype domains unfold fully reversibly, even when the conserved structural disulfide bridge is reduced. This has allowed us to probe the factors that limit the expression yield of soluble immunoglobulin domains in the reducing environment of the cytoplasm (intrabodies). The most important factor is thermodynamic stability, and there is an excellent quantitative correlation between stability and yield. Surprisingly, an unprocessed N-terminal methionine residue can severely compromise VL stability, but this problem can be overcome by changing the amino acid following the initiator methionine residue. Transcription from the strong T7 promoter does not increase the amount of soluble material over that obtained from the tetA promoter, but large amounts of inclusions bodies can be obtained. Elevated temperature shifts protein from a productive folding pathway to aggregation. The structural disulfide bridge does not form in the cytoplasm, but the two consensus cysteine residues can be quantitatively oxidized in vitro. In summary, stability engineering provides a plannable route to the high-yield cytoplasmic expression of functional intrabody domains. Study holds ProTherm entries: 6068, 6069, 6070, 6071, 6072, 6073, 6074, 6075, 6076 Extra Details: additive : EDTA(20 mM),oxidized intrabodies; recombinant expression; VL domain;,protein stability; protein engineering

Submission Details

ID: vkjsHhys3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:31 p.m.

Version: 1

Publication Details
Ohage E;Steipe B,J. Mol. Biol. (1999) Intrabody construction and expression. I. The critical role of VL domain stability. PMID:10518947
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)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Ig gamma-1 chain C region secreted form P01868 IGHG1_MOUSE
100.0 Ig gamma-1 chain C region secreted form P01869 IGH1M_MOUSE