A method for facile production of variable lymphocyte receptors using SHuffle Escherichia coli

Abstract

Variable lymphocyte receptors (VLRs) are the antigen receptors of jawless vertebrates such as lamprey. VLRs are of growing biotechnological interest for their ability to bind certain antigenic targets with higher affinity than traditional immunoglobulins. However, VLRs are disulfide-bonded proteins that are often challenging to produce requiring genetic modifications, fusion partners, non-scalable host cell lines or inclusion body formation and refolding. As a potential VLR expression platform option, the SHuffle Escherichia coli strain has been genetically altered to allow cytoplasmic disulfide bond formation by mutations to thioredoxin reductase (trxB) and glutathione reductase (gor) to create an oxidative cytoplasm. Furthermore, the SHuffle strain expresses disulfide bond isomerase DsbC in the cytoplasm to promote correct disulfide bond pairing. Here, we demonstrate that the SHuffle strain can produce high yield VLRs with titers ranging from 2 to 32 mg of VLR per liter of SHuffle culture. Three VLRs (P1C10, RBC36, VLRA.R2.1) were expressed in SHuffle E. coli and the products were compared directly to those generated using the Rosetta E. coli strain. All VLRs were validated for correct sequence, purity, and activity. For all VLRs, SHuffle E. coli produced 2-9 times more soluble VLRs than Rosetta E. coli. Furthermore, the soluble protein fraction was 2-6 times greater in SHuffle E. coli than Rosetta E. coli for all VLRs. Overall, these results suggest that the E. coli SHuffle strain is a convenient and effective expression system for producing large amounts of VLRs.

Keywords: SHuffle E. Coli; bacterial protein expression; disulfide bonds; variable lymphocyte receptors.

FIGURE 1

Variable lymphocyte receptors expression constructs in pET vectors with T7 promoters. (a) NT‐6xHis constructs were designed with a 6xHis tag and tobacco etch virus (TEV) cleavage site at the N‐terminus of the VLR. (b) CT‐6xHis constructs were designed with a 6xHis tag at the C‐terminus of the VLR.

FIGURE 2

Comparison of soluble variable lymphocyte receptor (VLR) yields using the SHuffle and Rosetta bacterial strains. (a–f) Western blot comparison for 6 VLR constructs. Bacterial lysates from Rosetta and SHuffle cultures were separated into insoluble and soluble fractions via centrifugation. Samples were run on reduced 4%–12% Bis‐Tris gels, transferred to a nitrocellulose membrane, and probed with anti‐6xHis antibody. (g–i) Densitometric quantification of the of total soluble VLR. Data were normalized to Rosetta NT‐6xHis for each VLR. (j–l) Densitometric quantification of the fraction soluble was assessed by dividing the total soluble material by the sum of the insoluble and soluble material. Data in all panels represent the mean and standard deviation of three independent bacterial transformants (n = 3). Data are expressed as mean ± SD and statistical significance was determined by a one‐way ANOVA with Tukey's post‐hoc test. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, all comparisons with no p‐value indicated were not statistically significant.

FIGURE 3

Reducing sodium dodecyl‐sulfate polyacrylamide gel electrophoresis assessment of purified, SHuffle‐produced variable lymphocyte receptors (VLRs). Ni‐NTA‐purified VLR preparations were resolved on a reducing 4%–12% bis‐Tris gel and visualized by Coomassie staining. Purified VLRs were loaded in duplicate lanes as depicted on the top of the gel. Molecular weight standards were loaded in lanes 1 and 14. Theoretical molecular weights for each construct are noted at the top of the gel.

FIGURE 4

Analysis of purified SHuffle‐produced variable lymphocyte receptors (VLRs) by size exclusion chromatography (SEC). (a). The purity for each VLR construct was assessed by SEC by integrating the largest peak and comparing to the total area under the elution curve in panels (b)–(d). (b–d) Purified VLR preparations were separated on a Superdex 200 Increase 10/300 GL column. The SEC chromatograms for each VLR were overlaid with that for the molecular weight standard to compare the peak retention times relative to the standard.

FIGURE 5

bEND.3 ECM ELISA to confirm activity of P1C10 variable lymphocyte receptors (VLRs). P1C10 NT‐6xHis and P1C10 CT‐6xHis binding was assayed at saturating concentrations with decellularized bEnd.3 ECM. RBC36 NT‐6xHis was used as a non‐binding control. Data represent the mean ± SD of assay replicates (n = 4). Statistical significance was determined by a one‐way ANOVA with Tukey's post‐hoc test. *** represents p < 0.001 compared to both NT and CT P1C10 6xHis, all comparisons with no p value present were not statistically significant.

FIGURE 6

Isothermal titration calorimetry (ITC) with SHuffle‐produced RBC36 and VLRA.R2.1 and their known binding partners to confirm activity. (a) H‐trisaccharide was titrated into TEV‐cleaved RBC36 (no 6xHis tag). (b) ITC data was fit to a nonlinear least squares binding model producing a K _D of 2.6 μM for RBC36. (c) Hen egg lysozyme (HEL) was titrated into TEV‐cleaved VLRA.R2.1 (no 6xHis tag). (d) ITC data was fit to a nonlinear least squares binding model producing a K _D of 2 nM for VLRA.R2.1.