Synthesis of [11C]interleukin 8 using a cell-free translation system and l-[11C]methionine
Introduction
Biological research is moving from the gathering of genomic sequences to protein expression for analysis of sequence function and utility. In accordance with this sequence-to-function paradigm shift, there have been great advances in biotechnology and bioengineering that enable the preparation of large quantities of numerous active proteins using simple yet sophisticated approaches [1], [2]. These technologies are rapidly spreading not only in many research fields, such as chemical biology, structural biology and pharmaceutical biology, but also in drug discovery and development. Especially, pharmaceutical peptides and proteins, including biotechnology-derived antibody, are becoming increasingly important as source of major new drugs [3], [4].
In addition to these advances in biotechnology and its applications, molecular imaging has come to be regarded as a key method for characterization of biomolecular functions and roles in vivo. Basic research and technology on molecular imaging have made great advances in recent years, and their application to drug development is highly expected [5]. One of the most useful and valuable imaging modalities in molecular imaging is positron emission tomography (PET), which uses a compound labeled with a positron emitter radioisotope as imaging probe. Positron emission tomography has several advantages over other imaging modalities, particularly in sensitive and quantitative investigations of molecular functions and processes in vivo. In addition, PET has a wide range of imaging objects extending from small animals to humans [6]. The use of appropriate positron emitter-labeled compounds is critical for PET molecular imaging because the characteristics of each labeled probe can define specific imaging information. Considering recent advances in biopharmaceuticals, positron emitter labeling of biomolecules for PET imaging is increasingly becoming important.
To date, several methods for labeling peptides and proteins with positron-emitting nuclides have been reported [2], [7]. For instance, biomolecules can be labeled by conjugation with a prosthetic group containing a positron emitter, such as N-succinimidyl-4-[18F]fluorobenzonate through acylation reaction, [18F]maleimide through formation of a thioether bond and [18F]fluoroalkyne through the click reaction [8]. Conjugation of a metal chelator, such as 1,4,7,10-tetraazacyclodocane-1,4,7,10-tetraacetic acid), with biomolecules followed by chelating of a positron emitter metal, such as 64Cu or 68Ga, also affords radiolabeled products [9], [10]. In addition, biomolecules can be directly radiolabeled with iodine-124 in the presence of an oxidizing agent, such as Iodogen or chloramine T [11]. Essentially, all of these conventional labeling methods are used to directly or indirectly attach a radioisotope to peptides or proteins based on a chemical reaction.
In this article, we propose a novel approach for synthesis of positron emitter-labeled proteins and peptides based on a biotechnology method. We focused on a cell-free protein synthesis system for preparation of radiolabeled peptides and proteins. The principle of our approach is to combine a cell-free system with a positron emitter-labeled amino acid. One of the most refined cell-free protein synthesis system is the PURE system, whose components are completely reconstituted purified essential elements from Escherichia coli with the composition of each component arbitrarily determined [12]. According to our approach, we chose interleukin 8 (IL-8) as an example to synthesize a radiolabeled chemokine using a commercially available PURESYSTEM and [11C]MET [13], [14]. Interleukin 8, a chemotactic factor that attracts neutrophils, basophils and T-cells, but not monocytes, and is involved in neutrophils activation, is released from several types of cells in response to inflammatory stimulus [15]. Since neutrophils express IL-8 receptors on their surface, positron emitter-labeled IL-8 would have the potential to be a PET probe for inflammation imaging. In fact, 99mTc- and 131I-labeled IL-8 is used for detection of infection and inflammation in clinical research [16], [17]. In this study, we investigated the possible use of a cell-free protein synthesis system for preparation of [11C]IL-8 as a PET probe.
Section snippets
Synthesis of IL-8 using a noncellular in vitro protein synthesis system
We synthesized recombinant human IL-8 protein in a cell-free system as described previously [1]. Plasmid DNA used for the synthesis of IL-8 was kindly gifted by Dr. A. Ametani (Post Genome Institute Co. Ltd.). The polymerase chain reaction (PCR) product used for the synthesis was prepared in our laboratory by two-step PCR method. In brief, in the first PCR step, we amplified cDNA using IL-8 specific primers with adaptor sequences at the 5′ terminus. Regulatory sequences, such as T7 promoter,
Results and discussion
Nonradiolabeled IL-8 was successfully synthesized using a cell-free translation system (PURESYSTEM), which is a unique reconstituted system consisting of a set of independently prepared enzymes, translation factors, ribosome, ATP, tRNA and amino acids necessary for protein synthesis [12]. As such, the composition of each component of this system can therefore be arbitrarily determined. This characteristic has a great advantage because the components of amino acids necessary for protein
Conclusions
In summary, we investigated the use of a cell-free protein synthesis system for preparation of positron emitter-labeled protein. [11C]IL-8 was synthesized using commercially available PURESYSTEM and [11C]MET as an example. The in vitro synthesis proceeded very smoothly, and maximum radioactivity of [11C]IL-8 was obtained within a short reaction time (20 min). In addition, purification of [11C]IL-8 could be achieved easily by a simple cation exchange and ultrafiltration system, resulting in
Acknowledgments
The authors appreciate Post Genome Institute Co. Ltd. (Japan) for giving precise technical information on PURESYSTEM. This work was supported by Grants-in-Aid for scientific research (nos. 21390171 and 21650088) from the Japan Society of Promotion of Science and the Ministry of Education, Culture, Sports, Science, and Technology in Japan. The authors also thank J. Tsubata, M. Kato and T. Ohnuki for their assistance.
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