Cell-free production of Gaussia princeps luciferase – antibody fragment bioconjugates for ex vivo detection of tumor cells

https://doi.org/10.1016/j.bbrc.2009.10.087Get rights and content

Abstract

Antibody fragments (scFvs) fused to luciferase reporter proteins have been used as highly sensitive optical imaging probes. Gaussia princeps luciferase (GLuc) is an attractive choice for a reporter protein because it is small and bright and does not require ATP to stimulate bioluminescence-producing reactions. Both GLuc and scFv proteins contain multiple disulfide bonds, and consequently the production of active and properly folded GLuc–scFv fusions is challenging. We therefore produced both proteins individually in active form, followed by covalent coupling to produce the intended conjugate.

We used an Escherichia coli-based cell-free protein synthesis (CFPS) platform to produce GLuc and scFv proteins containing non-natural amino acids (nnAAs) for subsequent conjugation by azide–alkyne click chemistry. GLuc mutants with exposed alkyne reactive groups were produced by global replacement of methionine residues in CFPS. Antibody fragment scFvs contained a single exposed azide group using a scheme for site-specific incorporation of tyrosine analogs. Incorporation of tyrosine analogs at specific sites in proteins was performed using an engineered orthogonal tRNA–tRNA synthetase pair from an archaebacterium. The unique azide and alkyne side chains in GLuc and the antibody fragment scFv facilitated conjugation by click chemistry. GLuc–scFv conjugates were shown to differentiate between cells expressing a surface target of the scFv and cells that did not carry this marker.

Introduction

Antibody fragments (single chain antibody Fv fragments, or scFvs) specific for unique cell surface markers can be used to differentiate cells that bear this marker (e.g. tumor cells) from other cells. Fusion of a luciferase reporter protein to antibody fragments allows highly sensitive detection of certain tumors in vivo and ex vivo[1], [2]. Firefly luciferase (FLuc) from Photinus pyralis and Renilla luciferase (RLuc) from Renilla reniformis are two extensively studied luciferases [3], [4]. The Gaussia princeps luciferase (GLuc) is a more attractive choice for a reporter protein because it is small, bright, and ATP-independent [2], [5]. GLuc and RLuc catalyze the oxidation of coelenterazine to coelenteramide accompanied by the emission of light. FLuc, RLuc and GLuc have been used in numerous in vitro and in vivo applications as reporter proteins [6], [7], [8], and luciferase and antibody fragment fusion proteins have been produced as reagents for detection of specific antigens for in vivo and in vitro imaging applications [1], [2], [9].

However, production of complex disulfide bonded proteins such as scFvs as well as GLuc using recombinant expression systems is challenging and production of properly folded and active bi-functional GLuc–scFv fusions is even harder. It is therefore desirable to produce both proteins separately in active form, followed by covalent coupling to produce the desired conjugate. Incorporation of non-natural amino acids (nnAAs) in proteins followed by direct linkage using azide–alkyne click chemistry [10], [11] is an attractive option since these reactions are efficient and can be performed under physiological conditions.

Our lab has developed a cell-free protein synthesis (CFPS) platform that facilitates incorporation of azide- and alkyne-containing nnAAs in proteins by adopting two different schemes; site-specific incorporation of tyrosine analogs [12], [13] as well as global replacement of methionine analogs [14]. Site-specific incorporation offers greater control and flexibility since a non-natural amino acid can be introduced at any desired site in a protein. The open cell-free system facilitates addition of optimal amounts of the orthogonal components, the tRNA and synthetase pair, which are required for site-specific incorporation of nnAAs in proteins [15]. Alternatively, the global replacement strategy mentioned earlier can provide higher yields of proteins since no orthogonal components are required and it appears that the methionine analogs are incorporated more efficiently [14]. However, the use of this method is limited to proteins where mutation of all methionine residues is not deleterious to protein folding or function. CFPS is also well suited for producing proteins containing methionine analogs since the absence of the cell wall barriers allows greater control over the concentrations of both methionine and the nnAA.

Cell-free protein synthesis has been successfully used to produce GLuc, scFv fragments, and other disulfide bonded proteins in soluble and active form with high yields [16], [17], [18], [19], [20]. We previously reported the cell-free production of GLuc mutants containing the methionine analogs azidohomoalanine (AHA) and homopropargylglycine (HPG) (Fig. 1A) [21]. The GLuc (HPG) mutant exhibited prolonged bioluminescence with an approximately 3-fold longer luminescence half-life as compared to the wild-type enzyme while retaining two-thirds of the wild-type specific activity. Further examination led to the identification of GLuc mutants containing methionine-to-leucine mutations at two critical positions, resulting in even higher luminescence half-lives and specific activities similar to wild-type. We also attached 5 kDa azide–PEG (polyethylene glycol) to each of the four HPG residues, suggesting that all four methionines in the native GLuc sequence are surface exposed and accessible for conjugation.

Here we demonstrate the ability of Gaussia luciferase – antibody fragment bioconjugates to detect cells bearing a unique surface marker; specifically, an interaction between a mouse B cell lymphoma tumor idiotype scFv and an anti-idiotype antibody [22] expressed as a cell-surface immunoglobulin (Fig. 2). The tumor idiotype scFv is produced as a fusion with the Escherichia coli IM9 protein, which has been shown to improve cell-free production of soluble scFv fusion proteins [18], [23]. The IM9 domain is designed to contain a site for the incorporation of tyrosine analogs at position 28, which is in a surface-exposed loop region (Fig. 1B). We produce GLuc–IM9scFv conjugates by first incorporating p-azido-l-phenylalanine (AZF, Fig. 1A) in the IM9scFv fusion protein and by replacing methionine residues in GLuc with HPG, followed by conjugation using Cu(I) catalyzed click chemistry. We constructed a mouse cell line that stably expresses surface anti-idiotype antibody, which is known to bind the tumor idiotype scFv [22]. The GLuc–IM9scFv conjugates were successfully used in an in vitro assay to differentiate between populations of cells expressing the anti-Id from cells that did not. Thus, we demonstrate the feasibility of using CFPS for the production of GLuc and scFv species that can be directly converted into bioconjugates capable of detecting cells that bear unique surface markers.

Section snippets

Experimental procedures

Plasmids for production of GLuc and IM9scFv. Plasmid pET24–AG1–GLuc–6H, described in [17] was used for cell-free production of GLuc containing HPG. It encodes amino acids 1–168 of the natural secreted protein sequence with the 17 amino acid signal sequence omitted and has been extended to encode an N-terminal methionine and a His6 sequence at the C-terminus (Fig. 1C).

Plasmid pY71-IM9(28TAG)-(38C13)scFv was constructed for expression of the IM9scFv fusion protein (Fig. 1D) which contains:

  • (1)

    the

Production and purification of proteins for conjugation

We previously reported that incorporation of the methionine analog HPG in GLuc using CFPS provided mutants with prolonged bioluminescence [21]. Incorporation of HPG only reduced GLuc specific activity by about a third while increasing the luminescence half-life from 1.3 min to 4 min. GLuc (HPG) was produced by CFPS with high yields (∼300–400 μg/mL) and purified using Ni–NTA chromatography. IM9scFv was produced using CFPS with the engineered orthogonal tRNA and tRNA synthetase pair from M.

Conclusions

The cell-free protein synthesis platform is well suited for the production of complex disulfide bonded proteins like GLuc and IM9scFv fusion proteins. The open cell-free system also allows for efficient production of these proteins with azide- and alkyne-containing nnAAs for conjugation. Interestingly, the use of a global replacement strategy to incorporate HPG residues in place of methionine in GLuc provided a mutant with prolonged bioluminescence as compared to the wild-type enzyme, making it

Acknowledgments

This work was supported in part by a grant from the Leukemia and Lymphoma Society.

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