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  • br Conclusion In this study we have shown the

    2020-11-24


    Conclusion In this study, we have shown the construction, expression and purification of CPG2 fused to the HIV-1 TAT peptide (TAT–CPG2). We have demonstrated for the first time that TAT-CPG2 in both native and denatured forms could be efficiently transduced into the HepG2 cells. Also, we have provided evidences for the enzyme activity of transduced TAT-CPG2 fusion protein. We assume that transduced CPG2 converts MTX to the non-toxic metabolites, which prevents the cell proliferation suppression and the oxidative stress caused by MTX. However, further investigations especially in vivo studies are required to elucidate the involved cellular mechanisms in depth. Our success in the protein transduction of TAT-CPG2 may provide a new strategy for protecting against cell toxicity resulting from MTX in various organs. Therefore, we propose that the TAT-CPG2 fusion protein could be used as an alternative to the gene-directed enzyme prodrug therapy.
    Introduction Carboxypeptidase G2 (CPG2) is a 42 kDa, zinc-dependent metalloenzyme from Pseudomonas that cleaves the glutamic Otilonium Bromide moiety from folic acid and its analogues. CPG2 is currently being exploited in a rescue therapy following high-doses of the highly cytotoxic drug methotrexate (MTX), commonly used in the treatment of cancer and autoimmune diseases, to metabolise the drug into two non-toxic metabolites: 2,4-diamino-N [10]-methylpteroic acid (DAMPA) and glutamate (Fig. 1A) [1], [2]. CPG2 has also played a key role in the development of antibody directed enzyme pro-drug therapy (ADEPT, see Fig. 1B and caption for full description) [3], an anti-cancer therapy aimed at limiting the action of cytotoxic drugs to tumour sites, thus amplifying their selectivity and effectiveness and diminishing the lethal effects on normal tissue [4]. It is envisaged that CPG2 will be used to generate active drugs (i.e benzoic acid mustard drugs) from a variety of glutamated pro-drugs (i.e glutamated benzoic acid mustard pro-drugs). CPG2 is ideal for use in ADEPT because it has no mammalian homologue, thus no endogenous enzymes would act on a pro-drug specific for CPG2, and being a bacterial enzyme has the advantage of enhanced kinetics with substrate turnover [5]. Due to its therapeutic applications in the treatment of cancer and autoimmune diseases, a robust understanding of the molecular determinants of CPG2 activity is of great interest for further development of therapeutics and applications. However, apart from an X-ray crystal structure of unliganded CPG2 reported by Rowsell and co-workers in 1997 [6] (PDB ID: 1CG2), our current working understanding of the molecular basis of CPG2 activity is largely based on sequence/structural homology and molecular modelling [7]. As shown in Fig. 1C, the crystal structure revealed a two-domain architecture for CPG2 consisting of a non-contiguous catalytic domain (Fig. 1D) and a dimerization domain thought to stabilize CPG2 homodimer formation. While the crystal structure informed early rational drug design efforts [6], [8], further attempts at co-crystallization with ligands, substrates or inhibitors have not been reported and very little progress has been made to establish key features of CPG2 such as the location of the active site, the presence of additional ligand-binding sites, stability, oligomeric state, and the molecular basis of activity. This lack of progress in CPG2 structural biology may be due (in part) to difficultly in formation of crystals for the ligand-bound enzyme. To take the next important steps toward characterizing CPG2, we wished to develop a robust method of making soluble, active enzyme in high yield with ready incorporation of a range of labels required for solution-state structural characterization. To date, CPG2 has been obtained either from the native strain Pseudomonas sp. RS 16 [9], or via expression using the Escherichia coli (E. coli) expression system [10] either in very low yield (100-fold lower than Pseudomonas) [11] or in an insoluble form requiring extensive unfolding and refolding steps and often resulting in low yields of active protein [12], [13]. The work presented here describes the first high-yield (250 mg L−1) recombinant expression (and purification) of soluble and active CPG2 using the E. coli expression system, achieved in part by removal of the N-terminal 22-residue signal peptide. This protocol was used to produce the full-length enzyme, as well as protein fragments corresponding to the individual catalytic and dimerization domains. A significantly truncated version of the catalytic domain was also designed and produced to narrow down the minimal requirements for CPG2 function. The activity and stability of each construct was characterised. The expression protocol was readily extended to the preparation of isotopically 2H/15N/13C-labelled CPG2 proteins (at yields of up to 109 mg L−1) for preliminary NMR analyses. NMR data were collected for all proteins, including the 42 kDa full-length enzyme, and an optimal candidate for future structure/binding studies was identified.