An improved purification procedure for cyclosporin synthetase
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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Cited by (5)
Rational biosynthetic approaches for the production of new-to-nature compounds in fungi
2016, Fungal Genetics and BiologyCitation Excerpt :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)).
Biosynthesis of cyclosporins and other natural peptidyl prolyl cis/trans isomerase inhibitors
2015, Biochimica et Biophysica Acta - General SubjectsCitation Excerpt :This in vitro approach avoids the sometimes inconsistent secondary metabolism of the fungus and the resultant inconsistent cyclosporin production observed. We have described purification methods for CySyn that result in almost homogenous preparations suitable for such an in vitro approach to the biosynthesis of desired CsA analogues [58,112]. Using suboptimal incubation temperature and long incubation times, we were able to produce μg-amounts of CsA analogues [59].
Cyclosporin A - A review on fermentative production, downstream processing and pharmacological applications
2011, Biotechnology AdvancesCitation Excerpt :Schorgendorfer et al. (1999) invented the method of altering the domains of CySyn to give modified enzyme with altered amino acid recognition specificity and to produce cyclosporin-like peptides or derivatives. Velkov et al. (2006) developed a practical and reliable method suited to large-scale processing to isolate CySyn for in vitro biosynthesis of cyclosporins. They reported that a sequence of chromatographic steps including ammonium sulfate precipitation, gel filtration, hydrophobic interaction chromatography and anion exchange chromatography yielded an electrophoretically homogeneous CySyn preparation.
Characterization of the N-methyltransferase activities of the multifunctional polypeptide cyclosporin synthetase
2011, Chemistry and BiologyCitation Excerpt :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).
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Present address: Department of Medicinal Chemistry, Victorian College of Pharmacy, Monash University, 381 Royal Parade, Parkville 3052 Vic., Australia.