Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli

The distance between the VH C terminus and the VL N terminus in Fab X-ray structures is about 3.5 nm, and a 15-residue linker spans it because one peptide unit is about 0.38 nm long. The flexible (Gly-Gly-Gly-Gly-Ser)3 linker, chosen for lacking a propensity for ordered secondary structure and for not interfering with domain folding, joins the VH carboxyl terminus to the VL amino terminus in the anti-digoxin 26-10 single-chain Fv (sFv).

An sFv built as prolyl-VH-(Gly4Ser)3-VL from the anti-digoxin monoclonal antibody 26-10 variable regions forms a single 248-residue polypeptide of Mr 26,354 that binds digoxin after expression in Escherichia coli, isolation and renaturation. Its association constant for digoxin by Scatchard analysis is (3.2 ± 0.9) × 10^7 M-1, about 6-fold lower than the (1.9 ± 0.2) × 10^8 M-1 of 26-10 Fab, measured in 0.01 M sodium acetate pH 5.5 with 0.25 M urea.

The 26-10 Fab has a lower Ka for digoxin in 0.01 M sodium acetate pH 5.5 than in 0.15 M NaCl/0.05 M potassium phosphate pH 7.0/0.03% NaN3 (3.3 × 10^9 M-1) so the Ka of the 26-10 sFv measured in acetate buffer may be similarly reduced by the buffer.

The 26-10 sFv with a (Gly4Ser)3 linker recognizes digoxin and related cardiac glycosides with a specificity profile similar to the 26-10 Fab, and is slightly more specific for digoxin. In the radioimmunoassay normalized to digoxin, the sFv needs 7.3-fold more digoxigenin and 150-fold more ouabain than digoxin for 50% inhibition of 125I-digoxin binding, against 1.2-fold and 15-fold for the Fab.

Connecting VH and VL through the 15-residue (Gly4Ser)3 linker is proposed to substitute for the constant-region contacts of the Fab and to reduce refolding problems of two-chain species, such as incorrect domain pairing or aggregation. In vitro recombination of 26-10 H and L chains gave a low yield of antibody with significantly reduced digoxin affinity.

Fusing the 26-10 sFv to a modified trp LE leader (MLE) as MLE-Asp-Pro-VH-(Gly4Ser)3-VL, under a synthetic trp promoter-operator in E. coli JM83 induced with indoleacrylic acid, gives high expression in which the fusion protein accumulates as insoluble granules. Acid cleavage of the engineered Asp-Pro bond in 6 M guanidine hydrochloride/acetic acid pH 2.5 at 37°C for 96 hr then releases sFv with an N-terminal proline.

A 744-base-pair sFv gene designed with E. coli preferred codons is assembled from synthetic oligonucleotides, with VH from 46 overlapping 15-base oligonucleotides, VL from 12 synthetic polynucleotides of 33 to 88 base pairs, and the (Gly4Ser)3 linker from two polynucleotides spanning the Sac I and Aat II sites.

The 26-10 sFv remains soluble when refolded by dilution to below 10 µg/ml in 0.01 M sodium acetate pH 5.5 and dialysis, whereas substitution of 0.15 M NaCl/0.05 M potassium phosphate pH 7.0/0.03% NaN3 causes precipitation. Adding 0.25 M urea to the pH 5.5 acetate buffer raises sFv activity by an order of magnitude without a change in Ka, and active binding species then represent 22% of sFv and 43% of 26-10 Fab by active-site concentration.

Affinity chromatography on ouabain-amine-Sepharose purifies active 26-10 sFv from renatured protein, with 12.6% of the renatured protein recovered as active sFv, which is 4.7% relative to the MLE-sFv fusion granules. The sFv bound to ouabain-Sepharose is fully active with stoichiometric digoxin binding capacity, but a substantial fraction forms inactive aggregates on elution.