Recombinant expression of TLR5 proteins by ligand supplementation and a leucine-rich repeat hybrid technique

Abstract

Vertebrate TLR5 directly binds bacterial flagellin proteins and activates innate immune responses against pathogenic flagellated bacteria. Structural and biochemical studies on the TLR5/flagellin interaction have been challenging due to the technical difficulty in obtaining active recombinant proteins of TLR5 ectodomain (TLR5-ECD). We recently succeeded in production of the N-terminal leucine rich repeats (LRRs) of Danio rerio (dr) TLR5-ECD in a hybrid with another LRR protein, hagfish variable lymphocyte receptor (VLR), and determined the crystal structure of its complex with flagellin D1-D2-D3 domains. Although the structure provides valuable information about the interaction, it remains to be revealed how the C-terminal region of TLR5-ECD contributes to the interaction. Here, we present two methods to obtain recombinant TLR5 proteins that contain the C-terminal region in a baculovirus expression system. First, production of biologically active full-length drTLR5-ECD was substantially enhanced by supplementation of expression culture with purified flagellin proteins. Second, we designed TLR5-VLR hybrids using an LRR hybrid technology by single and double LRR fusions and were able to express diverse regions of drTLR5-ECD, allowing us to detect a previously unidentified TLR5/flagellin interaction. The drTLR5-VLR hybrid technique was also successfully applied to human TLR5-ECD whose expression has been highly problematic. These alternative TLR5 expression strategies provide an opportunity to obtain a complete view of the TLR5/flagellin interaction and can be applied to other LRR proteins.

Fig. 1

^TN-x^V (A, B) and ^VTx-C (C, D) hybrid designs that allow linkage of LRR fragments of TLR5 and VLR. (A) Schematic representation of ^TN-x^V hybrids. In ^TN-17^V as an example of ^TN-x^V hybrids, the TLR5 N-terminal region from the N-terminus to LRR17 (yellow in continuous lines) was fused with the VLR C-terminal region from LRR4 to the C-terminus (cyan in continuous lines) at the 10th consensus residue (asparagine in red). (B) Amino acid sequences of ^TN-x^V hybrids at a fusing LRR module. TLR5 and VLR sequences are highlighted in yellow and cyan, respectively. A consensus residue where fusion occurs is shown in red. (C) A schematic representation of ^VTx-C hybrids. In ^VT1-C as an example of ^VTx-C hybrids, the TLR5 C-terminal region from the LRR1 to the C-terminus (yellow in continuous lines) was fused with the VLR N-terminal region from the N-terminus to LRR2 (cyan in continuous lines) at the 10th consensus residue (asparagine in red). (D) Amino acid sequences of ^VTx-C hybrids in the fusing LRR module.

Fig. 2

Size-exclusion chromatography profiles of drTLR5-ECD (A), ^TN-14^V (B), and ^VT11-C (C). Expressed proteins were first purified by Ni-NTA and Strep-Tactin affinity chromatography. The resulting proteins were digested by thrombin and injected into a Superdex 200 16/600 size-exclusion chromatography column.

Fig. 3

Improved expression yield of functional drTLR5-ECD proteins by FliC supplementation. Different amounts of purified FliC proteins (70 μg, 200 μg, or 600 μg) were added into 1 L of drTLR5-ECD expression culture at ~24 h postinfection. At 48 h postinfection, culture medium that contains drTLR5-ECD and FliC was harvested and subjected to purification by Ni-NTA affinity chromatography. The resulting material was analyzed using a Superdex 200 10/300 column to estimate the yield of drTLR5-ECD/FliC complex (A). Peak fractions that correspond to the drTLR5-ECD/FliC complex were analyzed by SDS–PAGE, indicating the presence of both drTLR5-ECD and FliC in the peak (B). Peak height and width increased in a FliC dose-dependent manner, demonstrating that FliC supplementation is a crucial step to improve drTLR5-ECD protein yield.

Fig. 4

Native PAGE analysis of interaction between drTLR5-VLR hybrids and CBLB502. CBLB502 was added in molar excess compared to drTLR5-VLR hybrids in each reaction. CBLB502 completely shifted ^TN-12^V, ^TN-14^V, and ^TN-17^V proteins by forming complexes. Noticeably, only a fraction of the ^VT11-C protein was complexed with CBLB502, suggesting a weak interaction.