Secretory expression of thyroid hormone receptor using transgenic silkworms and its DNA binding activity

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Highlights

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    We purified a functional thyroid hormone receptor, a type of nuclear protein, using the transgenic silkworm secretory expression.

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    The purified recombinant thyroid hormone receptor showed a specific DNA binding activity.

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    A poly-brominated biphenyl compound partially inhibited the DNA binding activity of the thyroid hormone receptor.

Abstract

Silkworms are economically important insects that have the ability to produce large amounts of silk. They have mass breeding methods and silk glands, which are specialized tissues that secrete silk fibroin and sericin. Thus, the production of recombinant proteins in a transgenic silkworm system is a promising approach. We developed a silkworm, Bombyx mori, as a host expression insect for recombinant proteins and successfully produced different proteins including antibodies, glycoproteins, and membrane receptors. The thyroid hormone receptor (TR) is a regulatory factor for many physiological phenomena. It is a lipophilic protein that has DNA-binding and ligand-binding domains. Based on our previous experiences, it was inferred that the recombinant TR easily formed aggregates and precipitates which is potentially due to an unstructured hinge domain. We applied the silkworm expression system to produce mice TRβ1 that was fused with glutathione S-transferase. Using 160 larvae, the yield of the recombinant GST-TRβ was approximately 4 mg, and the purified GST-TRβ completely retained its physiological activity. Our results indicated that the recombinant TRβ was secreted extracellularly using the silk fibroin signal peptide sequence. Moreover, we found that the expression system of silkworms was applicable to nuclear proteins.

Introduction

The thyroid hormone receptor (TR) is a type of nuclear receptor, activated by the binding thyroid hormone (TH), that plays a critical role in the regulation of cellular energy homeostasis. TH and TR are also involved in the development of many organisms including the central nervous system [1]. Because the TR initiates gene transcription by associating with the other cofactors, it acts as a ligand-dependent transcription factor to affect the gene regulation. TR has attracted researchers from various fields, and numerous functional analyses have been conducted. Moreover, the structural alteration of TR was also investigated. TR has a few domain structures termed as A/B, C, D, and E domains. The two major domains are the DNA-binding (DBD, domain C) and ligand-binding domains (LBD, domain E). The DBD is involved in binding to specific DNA elements called thyroid hormone response elements (TRE). The LBD allows TR to bind to TH with high affinity. The D-domain of TR links the DBD to LBD and forms an unstructured hinge that facilitates movement between the DBD and LBD. The DBD and LBD were independently purified as recombinant proteins and 3D dimensional structures [2,3]. In contrast, there have been a few biochemical reports using recombinant proteins with full-length TRs. The cell-based analysis methods (for examples, reporter gene assay) have been mainly employed to investigate the physiological functions of TR because it has characteristics that allow it to be easily aggregated and precipitated at a low concentration in vitro. This problem (potentially due to an unstructured hinge domain) makes the recombinant TR difficult to express and purify. For the expression of recombinant proteins, various host systems have been developed such as Escherichia coli, mammalian and insect cell cultures, etc. In this study, we developed and reported the silkworm, Bombyx mori, as a host expression system for recombinant proteins [4]. Silkworms have a special tissue for silk fibroin and sericin secretion, namely the silk gland. The excellent secretory expression systems of silkworms have not only been applied to secretory proteins, but also to membrane and intracellular proteins [[5], [6], [7], [8], [9]]. We reported the silkworm secretory expression system of the TR. Because the secretory mechanism is a strict quality control system for proteins, we expected that the secreted proteins by the silk gland will rarely aggregate and precipitate. Our results showed that the purified full-length TR completely retained its physiological activity.

Section snippets

Vector construction

The preparation of the transfer vector for the secretory expression of the mice thyroid hormone receptor β1 (TRβ) gene in the transgenic silkworm was conducted using the piggyBac derived transfer vector as reported previously [5,10]. The DNA fragment coding signal peptides of the silkworm fibroin heavy chain [11,12], E.coli glutathione S-transferase (GST), and mice TRβ (Fig. 1) were synthesized using the custom oligonucleotide synthesis service (Eurofins Genomics). This DNA fragment (Fig. 1)

Expression and purification of GST-TRβ fusion protein

The tissue specific recombinant expression in the posterior silk gland using the fibroin promoter and yeast Gal4 system was reported [13]. In addition, the use of the silkworm silk fibroin signal peptide was effective for the silk gland secretion [11,12]. Fibroin was a major protein contained in the silk gland extracts with the target recombinant proteins and was easily removed by fibrosis. We employed these backgrounds to express the GST-TRβ. To confirm the expression of the GST-TRβ in the

Conclusion

Using TG silkworm, we successfully purified functional TRβ, which was easily aggregated and was difficult for functional expression in other expression systems. The Secretory expression of the recombinant proteins using silkworms was effective even for proteins that translocate into the nucleus. We also showed that the bromine-containing biphenyl, BP-6, attenuated the DNA binding activity of TRβ.

Funding information

This work was supported by grants from the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 18H03379 to N·K, and Ministry of Agriculture, Forestry and Fisheries of Japan (Research Project for Sericultural Revolution) to S.T. This work was also supported by a grant from the Gunma University Medical Innovation Project to N.K. and S.T.

Acknowledgements

We thank Professor Yusuke Inoue (Faculty of Science and Technology, Gunma University, Japan) for their great help with the gel shift assay.

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These authors contributed equally.

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