Efficient isolation of soluble intracellular single-chain antibodies using the twin-arginine translocation machinery

Abstract

One of the most commonly used recombinant antibody formats is the single-chain variable fragment (scFv) that consists of the antibody variable heavy chain connected to the variable light chain by a flexible linker. Since disulfide bonds are often necessary for scFv folding, it can be challenging to express scFvs in the reducing environment of the cytosol. Thus, we sought to develop a method for antigen-independent selection of scFvs that are stable in the reducing cytosol of bacteria. To this end, we applied a recently developed genetic selection for protein folding and solubility based on the quality control feature of the Escherichia coli twin-arginine translocation (Tat) pathway. This selection employs a tripartite sandwich fusion of a protein-of-interest with an N-terminal Tat-specific signal peptide and C-terminal TEM1 beta-lactamase, thereby coupling antibiotic resistance with Tat pathway export. Here, we adapted this assay to develop intrabody selection after Tat export (ISELATE), a high-throughput selection strategy for the identification of solubility-enhanced scFv sequences. Using ISELATE for three rounds of laboratory evolution, it was possible to evolve a soluble scFv from an insoluble parental sequence. We show also that ISELATE enables focusing of an scFv library in soluble sequence space before functional screening and thus can be used to increase the likelihood of finding functional intrabodies. Finally, the technique was used to screen a large repertoire of naïve scFvs for clones that conferred significant levels of soluble accumulation. Our results reveal that the Tat quality control mechanism can be harnessed for molecular evolution of scFvs that are soluble in the reducing cytoplasm of E. coli.

Figure 1. Ampicillin resistant phenotypes and scFv solubility

(a) Cartoon of a sandwich fusion of a protein-of-interest (POI) to an N-terminal Tat specific signal peptide (ssTorA) and a C-terminal TEM1 β-lactamase (Bla) (b) Spot plates of 5-μL aliquots of MC4100 and B1LK0 cells expressing ssTorA-scFv13-Bla variants (WT, R1, R2, R3, and R4; plus BL1 isolated for intracellular function) on LB media. Overnight cultures were diluted and spotted on plates supplemented with 100 μg/mL Amp. (c) Growth rate in liquid LB supplemented with 100 μg/mL Amp. Error bars represent the standard error of 6 independent trials.

Figure 2. Quantitative ampicillin resistance as an indicator of scFv solubility

(a) Spot plates of 5-μL aliquots of MC4100 cells expressing ssTorA-scFv13-Bla variants on LB media. Overnight cultures were diluted in successive 10-fold dilutions (top-to-bottom) and spotted on increasing concentrations of Amp (left-to-right). Step diagram of both the (b) MIC and (c) MBC resistance profiles plotted as Amp concentration vs. dilution factor. (d) Spot plates of 5- μL aliquots of MC4100 cells expressing ssTorA-V_L-Bla and ssTorA-V_H-Bla variants on LB media. Overnight cultures from a 96-well plate were diluted 10-fold and then in successive 100-fold dilutions (top-to-bottom) and spotted on increasing concentrations of Amp (left-to-right).

Figure 3. Confirmation of phenotype and counter-screening of false positives

(a) Spot plates of 5-μL aliquots of MC4100 cells expressing selected variants on LB media. Overnight cultures were diluted 10⁴-fold and spotted on 0, 50, 200 μg/mL Amp. Spots were identified by phenotype as true positives (+), false positives (×), or unconfirmed (-). Note that cultures were not normalized by 600 nm absorbance prior to spotting. (b) Plasmids isolated from eight true positives and two false positives were restriction digested. The true positives (+) showed the presence of full-length genes while the false positives (×) showed either the presence of a small fragment or no gene insertion.

Figure 4. Evolution of a soluble scFv13 variant

(a) Spot plates of 5-μL aliquots of MC4100 cells expressing ssTorA-scFv13-Bla variants on LB media. Overnight cultures were diluted in successive 10-fold dilutions (top-to-bottom) and spotted on increasing concentrations of Amp (left-to-right). Step diagram of both the (b) MIC and (c) MBC resistance profiles plotted as Amp concentration vs. dilution factor.

Figure 5. Activity of affinity-independent and affinity-dependent evolved scFv13s

(a) A Western blot of the soluble lysate from BL21(DE3) cells expressing the indicated scFv13s appended with a c-myc affinity tag. (b) Densitometry of bands from three separate Western blots on the soluble cell lysate reported as percent increase relative to wt (gray bars) and percent increase in ELISA signal relative to wt on β-gal plates (white bars). (c) AMEF 959 cells expressing scFv13 R4, WT, BL1 (selected as a positive during functional screen, +), and a clone BL2 selected as nonfunctional (-) plated on X-gal. (d) AMEF 959 cells expressing scFv13 mutants in liquid culture supplemented with X-gal. In vivo activity is measured as the percent increase in 595 nm absorbance relative to wt. Error bars represent the standard error of 6 independent trials. (e) Image of a representative 96-well plate β-gal assay; top lane corresponds to samples as in (d) and bottom lane corresponds to empty control wells for comparison.

Figure 6. Isolation of a soluble scFv from the Tomlinson I library

(a) Spot plates of 5-μL aliquots of three cultures of MC4100 cells expressing scFvI9 spotted next to three cultures expressing arbitrary scFvs from the naïve Tomlinson I library in pSALect on LB media. Overnight cultures were diluted in successive 10-fold dilutions (top-to-bottom) and spotted on increasing concentrations of Amp (left-to-right). (b) Western blot analysis of 5 μL and 15 μL of soluble lysate from BL21(DE3) cells expressing scFvI9 and eight naïve scFvs from the Tomlinson I library appended with a c-myc affinity tag. (c) Sequence of clone I9 with diversity encoded at 18 positions, indicated with Xs, in four CDRs.

Figure 7. Structure of scFv13

The structure of scFv13 heavy chain (right) and light chain (left) is shown with the ribbon structure in gray and the disulfide bonds in green. A hydrogen bond is shown with a dashed green line. The amino acid substitutions in variant T3 are indicated with red side chains.