An improved purification procedure for cyclosporin synthetase

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Abstract

We have developed expedient and reliable methods to isolate cyclosporin synthetase for in vitro biosynthesis of cyclosporins. We have examined enzyme purification strategies suited to large-scale processing and present a chromatographic sequence that serves as a pilot model for industrial scale preparation of cyclosporin synthetase from cyclosporin producing fungi. A chromatographic sequence consisting of ammonium sulfate precipitation  gel filtration  hydrophobic interaction chromatography  anion exchange chromatography, yielded an electrophoretically homogeneous cyclosporin synthetase preparation (Coomassie G-250 brilliant blue staining). Furthermore, a native polyacrylamide gel electrophoresis system was developed for the isolation of active cyclosporin synthetase enzyme from crude extracts of cyclosporin producing fungi. The environmental factors affecting enzyme stability and the continuity of the in vitro cyclosporin biosynthetic reaction-temperature, pH, and substrate depletion were assessed and manageable conditions have been defined for sustainable cyclosporin biosynthesis with enzyme isolates. Cyclosporin synthetase exhibited an optimal temperature range of 24–29 °C and a pH optimum of 7.6. The native enzyme displayed a pI of 5.7, as determined by isoelectric focusing. The industrial implementation of an in vitro biosynthetic approach could potentially prove useful for the production of important therapeutic cyclosporins which occur as only minor fermentation by-products.

Section snippets

Radioisotopes and chemicals

S-Adenosyl-l-[methyl-14C]methionine ([14C-methyl]AdoMet, 58 mCi/mmol) was purchased from New England Nuclear (Boston, MA, USA). Bmt, cyclosporin standards, and FK506 were kindly donated by Dr. R. Traber (Novartis Ltd., Basel, Switzerland), the fungal strains were kindly donated by Dr. M. Dreyfuss (Novartis Ltd., Basel, Switzerland). All other chemicals were of the highest purity commercially available.

Growth of the organism

The culture of T. inflatum was performed as previously described [20]. The mycelial mass was

Production of CySyn by T. inflatum

Tolypocladium inflatum was harvested at the various times during growth in submerged cultures and crude extracts were prepared from equal biomass samples (1 g) of lyophilized mycelium. The activity of CySyn in extracts was assayed; an increase in enzyme activity was observed concomitantly with biomass and peaked following four days of growth (Fig. 2A). Following a 10 day period, a decrease in mycelial biomass is seen due to extensive hyphal fragmentation and lysis. This event is undesirable when

Discussion

The large spectrum of pharmacological actions of CsA has placed great importance on the search for derivatives that possess specific properties without the adverse effects. However, the large-scale biotechnical production of CsA analogs has yet to be realized. Presently, industrial processes for large-scale CsA production involve a number of in vivo systems, predominantly submerged or solid state fermentation [35] or immobilized cells [36], [37]. The NRPS system, CySyn, responsible for the

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      Such assays, examining the substrate specificity of the cyclosporine synthetase (CySyn), were performed by Lawen, Traber and co-workers: Feeding sets of amino acids to isolated CySyn in a cell-free system resulted in the generation of new analogs, which could not be detected by PDB in vivo (Lawen and Traber, 1993; Lawen et al., 1994, 1991, 1989). Although there are optimized protocols for the purification even of large multidomain synthetases (e.g. for the 1.7 MDa CySyn (Velkov et al., 2006)), the isolation of required enzymes is still a challenging task, particularly if more than one enzyme is involved in the biosynthesis. Recently, Tang, Vederas and co-workers reconstituted the biosynthetic pathway of cladosporin in S. cerevisiae by heterologously expressing the type I PKSs Cla2 (highly reducing PKS (hr-PKS)) and Cla3 (non-reducing PKS (nr-PKS)).

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      Sinefungin was a generous gift from Eli Lilly Research Laboratories (Indianapolis, IN). These methods were performed as previously described (Velkov et al., 2006). N-MTase activity was assayed by monitoring the incorporation of [14C]-methyl groups from S-adenosyl-l-[methyl-14C]methionine into the amide nitrogens of CsA, according to Lawen et al. (1989) and Lawen and Zocher (1990).

    1

    Present address: Department of Medicinal Chemistry, Victorian College of Pharmacy, Monash University, 381 Royal Parade, Parkville 3052 Vic., Australia.

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