New method for immobilising diverse proteins onto cubic micro-protein polyhedrin crystals

Highlights

• Polyhedrin protein cubic crystals can be a stage to immobilise foreign protein.

• Several proteins were previously unable to be immobilised.

• This new method allowed the crystals to immobilise diverse proteins.

• This method will expand the application potential of polyhedrin protein crystals.

Abstract

Cypovirus is an insect virus that is encapsulated in stable cubic protein crystals composed of polyhedrin protein produced in virus-infected cells. Molecular technology developed over the last decade is now able to immobilise proteins of interest on polyhedrin crystals. Modified polyhedrin crystals can be used in cell cultures for implantation in animals and vaccines, among other applications. However, this technique does not work for some proteins. Here, we developed and tested an alternative approach for immobilising foreign proteins in polyhedrin crystals using a linker method; diverse proteins, such as fluorescent proteins, enzymes, antibodies, and streptavidin were successfully contained. The immobilised antibodies retained their binding activity on filter paper, implying their potential for new immunochromatography applications. Moreover, this immobilisation method allows enzymes to be collected from one reaction reagent and transferred to another reagent. These results demonstrate the potential of this immobilisation method and the likelihood of expanding the applications of polyhedrin crystals using this approach.

Introduction

Cytoplasmic polyhedrosis viruses produce polyhedra, which are robust protein crystals composed of polyhedrin protein encoded by the virus genome. The large amount of monomer polyhedrin that is produced subsequently self-assembles with virus particles in the cytoplasm of infected insect cells; thus, the virus particles are effectively encapsulated within the assembled polyhedrin crystals. The protein crystal protects the virus particles from severe environments for several years [[1], [2], [3]]; however, polyhedrin crystal can be dissolved in alkaline conditions, such as that of the caterpillar midgut. Dissolved crystals in the midgut release the virus particles, which in turn infects the midgut epithelium.

As revealed by analysis of the atomic structure of polyhedrin crystals, key contributors to the assembly of polyhedrin include a tyrosine cluster and N-terminal H1 α-helix domains [2]. Both structures provide denseness to the crystal, which is composed of an assembly of eight molecules of a polyhedrin homotetramer. The application of polyhedrin crystals for protection of foreign proteins has been studied, and immobilisation of green fluorescent protein (GFP) within polyhedrin crystals has been demonstrated using a partial sequence of viral protein 3 (VP3) as a VP3 tag [4]. In this method, the DNA of VP3-tagged GFP fusion protein and polyhedrin are transduced by virus vectors into Spodoptera frugiperda IPLB-Sf21AE (Sf21) insect culture cells. The doubly infected Sf21 cells co-express polyhedrin and VP3-tagged GFP and produce polyhedrin crystals containing GFP. In addition to the VP3 sequence, another tag sequence for immobilisation of foreign proteins in polyhedrin crystals has also been developed. The H1 α-helix domain at the N-terminal of the polyhedrin protein can be used as an H1 tag to immobilise foreign proteins within polyhedrin crystals, similar to VP3 [5]. These types of VP3 and H1 tags allow a large amount of protein to be immobilised within polyhedrin crystals, while retaining their activity [[5], [6], [7]]. In other words, this co-expression method extends the applications of the polyhedrin crystals. Polyhedrin protein crystals can easily adsorb onto a plastic culture dish or plate. Thus, it is possible that soluble or diffusible proteins immobilised on polyhedrin protein crystals can reside at specific positions on the culture vessel. Polyhedrin protein crystals can also be used in combination with other materials. For example, fibroblast growth factor-immobilised polyhedrin crystals mixed in collagen gel provide a base for three-dimensional culture of keratinocytes and melanocytes in vitro [8]. Further, a collagen sponge soaked in a cytokine-immobilised polyhedrin crystal suspension can be implanted into mice [[9], [10], [11]]. The polyhedrin crystals, as 1–5-μm cubes, are caught in the sponge and dissolved slowly by blood components. These implantation assays confirm that polyhedrin crystal is a biologically inert material.

A number of different types of proteins have been tested for immobilisation with this co-expression method using VP3 or H1. However, some proteins were unable to retain their activity after immobilisation. The cause of this loss of activity is unclear but may be due to the loss of the native structure of the target proteins through immobilisation, mediated by the VP3 or H1 tag. In addition, proteins of unknown sequence could not be transduced into co-expression systems for tagging on VP3 or H1. Furthermore, preparation using this method was unsuccessful for multimeric proteins, such as antibody or streptavidin, and cytotoxic protein was also not expressed in Sf21 cells, due to the toxicity of H1 tagged pierisin, for example [12]. To expand the application potential of polyhedrin crystals, we sought to develop a new method to immobilise these types of proteins. Herein, we present an alternative method to immobilise any protein on polyhedrin crystals without loss of activity. Briefly, recombinant protein was conjugated onto plain polyhedrin crystals prepared from single infected cells. This allowed diverse proteins to retain their activity following immobilisation, including fluorescent proteins, enzymes, streptavidin, and antibodies. We also demonstrated exploratory results that indicate the application potential of this method. Our results show that the proposed method is applicable to many proteins that cannot be immobilised by conventional co-expression methods.