The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.
With intensities 10(8)-10(10) times greater than other laboratory sources, X-ray free-electron lasers are currently opening up new frontiers across many areas of science. In this Review we describe how these unconventional lasers work, discuss the range of new sources being developed worldwide, and consider how such X-ray sources may develop over the coming years
A technique is proposed to generate attosecond pulse trains of radiation from a Free-Electron Laser amplifier. The optics-free technique synthesises a comb of longitudinal modes by applying a series of spatio-temporal shifts between the co-propagating radiation and electron bunch in the FEL. The modes may be phase-locked by modulating the electron beam energy at the mode spacing frequency. Three-dimensional simulations demonstrate the generation of a train of 400 as pulses at giga-watt power levels evenly spaced by 2.5 fs at a wavelength of 124Å. In the X-ray at wavelength 1.5Å, trains of 23 as pulses evenly spaced by 150 as and of peak power up to 6 GW are predicted.
For decades microbial exopolysaccharides have been invaluable ingredients in the food industry, as well as having many attractive pharmaceutical and chemical applications. Gellan gum is a comparatively new gum elaborated by the Gram-negative bacterium Sphingomonas paucimobilis. Although its physico-chemical properties have been well characterized, the ecology and physiology of Sphingomonas, and the factors influencing the fermentation process for production of this gum have received much less attention. This review focuses on the metabolism and the enzymic activity of this bacterium, as well as the factors that influence gellan production, including process temperature, pH, stirring rate, oxygen transfer, and composition of the production medium. Potential strategies for improving the production process are discussed in the context of processes for the production of other microbial biopolymers, particularly exopolysaccharides. In addition, the importance and potential utility of gellan lyases in modification of gellan and in other applications is critically evaluated.
A method is demonstrated that allows a planar wiggler high-gain Free-Electron-Laser (FEL) amplifier to lase so that the interaction with an odd harmonic of the radiation field dominates that of the fundamental. This harmonic lasing of the FEL is achieved by disrupting the electron interaction with the usually dominant fundamental while allowing that of a harmonic interaction to evolve unhindered. The disruption is achieved by a series of relative phase changes between the electrons and the ponderomotive potentials of both the fundamental and harmonic fields. Such phase changes are relatively easy to implement and some current FEL designs would require little or no structural modification to test the scheme.
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