Expression and purification of a native Thy1-single-chain variable fragment for use in molecular imaging

Abstract

Molecular imaging using singlechain variable fragments (scFv) of antibodies targeting cancer specific antigens have been considered a non-immunogenic approach for early diagnosis in the clinic. Usually, production of proteins is performed within Escherichia coli. Recombinant proteins are either expressed in E. coli cytoplasm as insoluble inclusion bodies, that often need cumbersome denaturation and refolding processes, or secreted toward the periplasm as soluble proteins that highly reduce the overall yield. However, production of active scFvs in their native form, without any heterologous fusion, is required for clinical applications. In this study, we expressed an anti-thymocyte differentiation antigen-scFv (Thy1-scFv) as a fusion protein with a N-terminal sequence including 3 × hexa-histidines, as purification tags, together with a Trx-tag and a S-tag for enhanced-solubility. Our strategy allowed to recover ~ 35% of Thy1-scFv in the soluble cytoplasmic fraction. An enterokinase cleavage site in between Thy1-scFv and the upstream tags was used to regenerate the protein with 97.7 ± 2.3% purity without any tags. Thy1-scFv showed functionality towards its target on flow cytometry assays. Finally, in vivo molecular imaging using Thy1-scFv conjugated to an ultrasound contrast agent (MB_Thy1-scFv) demonstrated signal enhancement on a transgenic pancreatic ductal adenocarcinoma (PDAC) mouse model (3.1 ± 1.2 a.u.) compared to non-targeted control (0.4 ± 0.4 a.u.) suggesting potential for PDAC early diagnosis. Overall, our strategy facilitates the expression and purification of Thy1-scFv while introducing its ability for diagnostic molecular imaging of pancreatic cancer. The presented methodology could be expanded to other important eukaryotic proteins for various applications, including but not limited to molecular imaging.

Figure 1

Recombinant protein checkpoints.

Figure 2

Schematic workflow showing the methodology employed for the construction, expression and purification of recombinant protein in its native form.

Figure 3

Construction and expression of recombinant Thy1-scFv proteins. (a) Schematic map showing the cloning pattern of pET-32b vector constructed to express recombinant Thy1-scFv in various formats (different number of hexa-histidine tags): pET32b-1XHis-scFv, pET32b-3XHis-scFv, and pET32b-5XHis-scFv. (b) Purity of protein based on the elution fractions after purification by affinity chromatography: Thy1-scFv formats resolved in 4–12% gradient SDS-PAGE. M protein molecular weight marker, IF insoluble fraction from cell lysate, SF soluble fraction from cell lysate, FT flow through, lanes 1–9: eluted fractions; arrows indicate the position of the tagged-Thy1-scFvs. The same methodology was applied for all Thy1-scFv formats and gels were processed in parallel. Dotted lines have been used to delineate different gels. Full-length gels are presented in Supplementary Figure S1.

Figure 4

Optimization of EK incubation time and concentrations from (a) 0.5U and 1U to (b) 2U and 8U, for tag removal of the recombinant Thy1-scFv protein. 4–12% gradient SDS-PAGE were processed in parallel.

Figure 5

Second step purification of Thy1-scFv after proteolysis. (a) SDS-PAGE showing pure Thy1-scFv. M protein molecular weight marker, FT flow through. (b). Mass spectrometry on Thy1-scFv flow through fraction.

Figure 6

Elution profile of Thy1-scFv on Ni–NTA IMAC. (a) IMAC1: tagged-Thy1-scFv is collected during the elution step. (b) IMAC2: native Thy1-scFv is collected in the flow through fraction. Black arrows represent the time of injection, and blue arrows indicate the peak and elution time of the protein of interest.

Figure 7

Binding efficiency of Thy1-scFv to MS1 cells.

Figure 8

In vivo USMI of Thy1 expression in transgenic mouse model with spontaneous PDAC. (a) Schematic illustration of the overall experiment. After localizing the tumor by US B-mode abdominal imaging, MB_non-targeted and MB_{Thy1-scFv xf}were successively tested for Thy1 binding in PDAC transgenic mice. Orange arrows delineate a pancreatic tumor. B-mode images were used as references to draw region of interest (ROIs) in differential targeted enhancement (dTE) images presented in (c). Scale bar 1 mm; (b) Quantitative bar graphs of in vivo dTE using targeted and non-targeted contrast agents in PDAC and healthy mice. **p < 0.03; ***p < 0.02; (c) Representative transverse dTE images showing stronger signal enhancement in PDAC (green ROI) after injection of MB_Thy1-scFv, and only low signal following injection of MB_non-targeted. Background signal was noted in adjacent normal pancreas (a yellow ROI was drawn to quantify imaging signal in adjacent non-PDAC tissue). Scale bar 1 mm; color coded scale is shown for USMI in arbitrary units (a.u.). (d) Corresponding hematoxylin–eosin-stained sample confirmed presence of PDAC in transgenic animals. Scale bar 500 μm.