Cell-Free System Stability

As discussed above, the purification and reconstitution of active PKSs from a variety of heterologous expression systems (including E. coli) are now feasible. Given the substantial tolerance of PKSs toward altered substrates and intermediates, it should therefore be possible to exploit this catalytic potential in a far more powerful way in cell-free systems than in intracellular systems. The primary limitations are with regard to the scale of synthesis. Attempts to stabilize and reuse the enzymes, in conjunction with the development of cheaper sources of natural and unnatural substrates and recycling systems for NADPH, should go a long way toward ameliorating this limitation. 

Endo and co-workers at Ehime University, Matsuyama, Japan, have led the development of the most promising eukaryotic cell-free system to date, based on wheat embryos. A significant advance made by this group was the development of pEU expression vectors that have overcome many of the difficulties associated with mRNA synthesis for translation in a eukaryotic system [8]. In addition to extensive optimization of reaction conditions that have seen improvements in protein synthesis rates, Endo and colleagues have improved wheat extract embryo preparation protocols to enhance the stability of these systems to a remarkable extent [9]. When coupled with the dialysis mode of reaction, Endo et al. were able to maintain translational activity in a coupled transcription/ translation wheat embryo reaction for 150 hours, producing 5 mg of enzymatically active protein per mb reaction mixture [10]. This again represents a serious alternative to in vivo methods of large-scale protein production. 

The great structural complexity encountered among sesquiterpenes implies that multiple cyclizations, shifts, and rearrangements of the parent cyclic intermediates may occur before stabilization to the final product, and in many instances several routes to the same sesquiterpene are feasible. Whether such reactions are concerted, take place sequentially on the enzyme surface, or involve discrete free intermediates is not yet generally known, and all three mechanistic alternatives, as well as multiple pathways, have been invoked to rationalize the same sesquiterpene structures. Because very few cell-free systems are available to examine these questions directly, and because of the difficulties associated with obtaining direct evidence of biosynthetic routes via in vivo tracer studies, hypothetic il pathways based mainly on chemical reasoning have been the rule (Roberts, 1972 Rucker, 1973 Anderson ef al., 1978). 

As indicated above, the antitumor activity of Epo B is based on its ability to bind to microtubules and to alter their intrinsic stability and dynamic properties. (For excellent reviews on microtubule structure and function see ref. 6 and 47-49.) Epothilones prevent the Ca or cold-induced depolymerization of pre-existing microtubule polymers in cell-free systems at the same time, they promote the polymerization of soluble tubulin into microtubule-like polymers under conditions that would normally destabilize microtubules.As demonstrated by kinetic experiments, epothilones inhibit the binding of taxol to microtubules in a competitive manner and they bind to the taxol binding site on p-tubulin with affinities that exceed (Epo B) or are comparable (Epo A) to taxol affinity likewise Epo B is a more potent tubulin-polymerizing agent than taxol or Epo Structural studies on... 

Fig. 1. Overview of the wheat germ cell-free translation system developed at our laboratory. The sequential establishment of the system started with stabilization, followed by integration with flexible plasmid of Ehime University (pEU) vector and finally high-throughput protein synthesis. (From ref. 21a, with permission from Elsevier.)...

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