Multiply mutated Gaussia luciferases provide prolonged and intense bioluminescence

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Abstract

Gaussia luciferase (GLuc) from the copepod Gaussia princeps is both the smallest and brightest known luciferase. GLuc catalyzes the oxidation of coelenterazine to produce an intense blue light but with a very short emission half-life. We report mutated GLucs with much longer luminescence half-lives that retain the same initial intensity as the wild-type enzyme. The GLuc variants were produced using cell-free protein synthesis to provide high yields and rapid production of fully active product as well as simple non-natural amino acid substitution. By incorporating homopropargylglycine and attaching PEG using azide–alkyne click reactions, we also show that the four methionines in GLuc are surface accessible. The mutants provide a significantly improved reporter protein for both in vivo and in vitro studies, and the successful non-natural amino acid incorporation and PEG attachment indicate the feasibility of producing useful bioconjugates using click attachment reactions.

Introduction

Bioluminescence imaging using luciferase enzymes has proven to be a useful tool for both in vivo and in vitro imaging in a variety of different applications [1], [2], [3], [4], [5], [6]. Firefly luciferase (FLuc) from Photinus pyralis and Renilla luciferase (RLuc) from Renilla reniformis are two of the most studied luciferases [7], [8], [9], [10]. FLuc catalyzes the oxidation of luciferin in the presence of the co-factor ATP and displays stable glow kinetics, whereas RLuc catalyzes the oxidation of coelenterazine in an ATP-independent manner while producing rapid flash kinetics [3]. More recently, Gaussia luciferase (GLuc) from the marine copepod Gaussia princeps has been shown to give much brighter luminescence than either FLuc or RLuc [3], [11]. Like RLuc, GLuc requires coelenterazine as a substrate and does not require ATP as a co-factor. In addition to its brighter signal, GLuc is also relatively stable due to its multiple disulfide bonds, and it is the smallest known luciferase, making it attractive as a fusion partner with nucleic acids or proteins for producing biomolecular probes and other useful conjugates. GLuc is naturally secreted in eukaryotic cells and has been successfully used for tumor imaging via antibody fusions [12] and as an ex vivo monitor of cellular growth, survival, and gene transduction in blood [3], [5]. However, more widespread use of GLuc in imaging applications has been hampered by: the enzyme’s blue emission peak (480 nm) which is rapidly attenuated by hemoglobin thereby limiting its in vivo efficacy, difficulties in obtaining high yields of active product, and flash kinetics of luminescence that decay rapidly within minutes. Several commercially available stabilizers (Invitrogen, Carlsbad, CA and Targeting Systems, El Cajon, CA) provide a more sustained luminescence signal but cause an approximately tenfold reduction in peak luminescence signal thereby significantly reducing the sensitivity of the reporter. Recently, Maguire et al. [13] reported a mutant GLuc with a more sustained luminescence signal. This mutant contains a single point mutation (M43I) that extends the half-life of light emission intensity from 2.4 min to 9.1 min when assayed in vitro using purified enzymes in the presence of the detergent, Triton X-100. However, the specific activity of this mutant is lower by approximately threefold.

Cell-free protein synthesis (CFPS) has been shown to facilitate high-level production of active GLuc [11]. The open nature of the cell-free system allows facile modification to the reaction environment, and the lack of intact cells provides for simpler purification procedures. The cell-free platform has been shown to be useful for the production of a variety of different proteins including: integral membrane proteins [14], virus-like particles [15], human transcription factors for nuclear reprogramming (in press), and disulfide bond containing proteins such as lymphokines [16]. For GLuc, the cell-free environment allows the introduction of an oxidizing glutathione buffering system and a protein disulfide isomerase; these enhance the formation of the disulfide bonds necessary for enzymatic activity. Due to the high specific activity and production yields observed for GLuc produced by CFPS, monitoring of luminescence is also easier since the activity can be assessed directly in the crude cell-free reaction product without the need for purification.

Incorporation of non-natural amino acids (nnAAs) containing azide and alkyne groups facilitates post-translational modifications and protein–protein conjugation by azide–alkyne click chemistry [17], [18], [19]. CFPS is particularly well suited for the introduction of nnAAs in proteins since it avoids potential pitfalls such as the toxicity of non-natural components and limitations in nnAA intracellular uptake.

In order to create GLuc-antibody fragment conjugates for tumor cell detection, we adopted a strategy for global replacement of the methionine residues in GLuc with azidohomoalanine (AHA) or homopropargylglycine (HPG) [18]. We observed that GLuc mutants containing AHA and HPG exhibit altered luminescence kinetics. By then mutating methionine residues to leucines, we identified two key mutations that give rise to a GLuc mutant with nearly full wild-type activity and with prolonged luminescence half-life relative to the M43I mutant reported by Maguire et al. [13]. We also demonstrate that all four methionine residues are surface accessible and indicate the feasibility of GLuc conjugation to fusion partners using click chemistry.

Section snippets

Materials and methods

Plasmids for production of GLuc. Plasmid pET24–AG1–GLuc–6H, described in [11] was used for cell-free production of GLuc. 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 (denoted as position (0) in Fig. 1) and a His6 sequence at the C-terminus. This same plasmid template was used for the production of GLuc containing the non-natural amino acids azidohomoalanine (AHA) and

Identification of a Gaussia luciferase mutant with sustained luminescence

In order to create bioconjugates with Gaussia luciferase (GLuc) for various applications where the GLuc would serve as a sensitive reporter molecule, we used the CFPS platform to incorporate the non-natural amino acids homopropargylglycine (HPG) and azidohomoalanine (AHA) (Fig. 1) using global replacement of the methionines as previously described by Strable et al. [18]. Both mutants were produced at high levels similar to the native enzyme using cell-free protein synthesis, and both mutants

Conclusion

We have identified a specific methionine to leucine mutation in Gaussia luciferase that confers stabilized light emission signal at the expense of a drop in specific activity, similar to a recent finding [13]. We have also identified an additional mutation that, in combination, further increases the light emission half-life while restoring the specific activity to nearly that of the wild-type enzyme. To our knowledge, this combination of mutations produces the most active GLuc with the longest

Acknowledgments

The authors thank Dr. Jianghong Rao and Dr. Zuyong Xia from the Stanford School of Medicine for assistance in obtaining emission spectra and Randall Lowe from the Chris Chidsey group in the Stanford Chemistry Department for providing the TTMA Cu(I) ligand.

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    These authors contributed equally.

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