Optimizing recombinant antibodies for intracellular function using hitchhiker-mediated survival selection

Abstract

The 'hitchhiker' mechanism of the bacterial twin-arginine translocation pathway has previously been adapted as a genetic selection for detecting pairwise protein interactions in the cytoplasm of living Escherichia coli cells. Here, we extended this method, called FLI-TRAP, for rapid isolation of intracellular antibodies (intrabodies) in the single-chain Fv format that possess superior traits simply by demanding bacterial growth on high concentrations of antibiotic. Following just a single round of survival-based enrichment using FLI-TRAP, variants of an intrabody against the dimerization domain of the yeast Gcn4p transcription factor were isolated having significantly greater intracellular stability that translated to yield enhancements of >10-fold. Likewise, an intrabody specific for the non-amyloid component region of α-synuclein was isolated that has ~8-fold improved antigen-binding affinity. Collectively, our results illustrate the potential of the FLI-TRAP method for intracellular stabilization and affinity maturation of intrabodies, all without the need for purification or immobilization of the antigen.

Keywords: antigen-binding affinity; directed evolution; intracellular antibody engineering; protein folding and stability; twin-arginine translocation.

Fig. 1.

Optimizing recombinant intrabodies using FLI-TRAP. Schematic representation of engineered assay for co-translocation of interacting receptor-ligand pairs via the Tat translocase. By simply demanding bacterial growth on high concentrations of antibiotic, the intracellular stability and/or antigen-binding affinity can be enhanced. The Tat signal peptide chosen was ssTorA, the reporter enzyme was Bla, the receptor was either scFv-GCN4(GLF) or NAC32, and the ligands were GCN4(7P14P) or α-syn(A53T).

Fig. 2.

Direct selection of improved scFv-GCN4 variants. (A) Representative spot titers for serially diluted Escherichia coli MC4100 cells co-expressing TatABC along with GCN4(7P14P)-Bla and one of the following: ssTorA-scFv-GCN4(GLF), the low-affinity ssTorA-scFv-GCN4(GFA) variant, or library-selected clones m1–m3 derived from scFv-GCN4(GLF). Overnight cultures were serially diluted in liquid LB and plated on LB agar supplemented with Carb. Maximal cell dilution that allowed growth is plotted versus Carb concentration. Arrow indicates Carb concentration depicted in the panel above the graph (500 μg/ml). (B) Western blot analysis of cytoplasmic (cyt) and periplasmic (per) fractions prepared from the same cells as in (A). An equivalent number of cells were loaded in each lane. Blots were probed with an anti-Bla antibody to detect GCN4(7P14P)-Bla and an anti-FLAG antibody to detect scFv-GCN4 intrabodies. Quality of fractions was confirmed by probing membranes with an anti-GroEL antibody.

Fig. 3.

Intracellular accumulation and antigen binding of scFv-GCN4 variants. (A) Western blot analysis of soluble and insoluble fractions from BL21(DE3) cells expressing scFv intrabodies without signal peptides from pET-28a. Clone m1, m2 and m3 were isolated using a single round of mutagenesis and FLI-TRAP selection. scFv-GCN4(GLF) was the starting sequence for the library, and scFv-GCN4(GFA) was a low-affinity derivative of scFv-GCN4(GLF) used here as a control. Samples were normalized by total protein concentration in the soluble fraction, and blot was probed with an anti-FLAG antibody. (B) ELISA data for binding of isolated clones to GCN4. Intrabodies were purified from cell lysate, and their binding to GCN4-coated ELISA plates was measured. Bound scFv intrabodies were detected with an anti-FLAG antibody. ELISA data are normalized to the signal for clone m3 at ∼5 μg/ml and expressed as the mean ± standard error of the mean of six biological replicates.

Fig. 4.

Direct selection of improved NAC32 variants. (A) Selective plating of Escherichia coli MC4100 cells co-expressing α-syn(A53T)-Bla with ssTorA-NAC32 or FLI-TRAP-selected NAC32 variants. Overnight cultures were serially diluted in liquid LB and plated on LB agar supplemented with Carb. Clone NAC32.R1 was derived from wt NAC32 following an initial round of error-prone PCR mutagenesis and selection on 50 μg/ml Carb. A second round of error-prone PCR mutagenesis, using NAC32.R1 as template, and selection on 100 μg/ml Carb was used to isolate NAC32.R2. (B) Antigen binding activity and (C) western blot analysis of NAC32 and FLI-TRAP-selected variants. NAC32 and its derivatives were expressed from plasmid pET-28a without the ssTorA signal peptide and with a C-terminal FLAG epitope tag. Binding activity in cell lysates was measured by ELISA with microtiter plates coated with α-syn(A53T) as antigen. Western blot analysis of soluble and insoluble fractions was according to standard protocols. Detection was with anti-FLAG antibodies. ELISA data are expressed as the mean ± standard error of the mean of six biological replicates.