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Protein crystallization is of strategic and commercial relevance in the post-genomic era because of its pivotal role in structural proteomics projects. Although protein structures are crucial for understanding the function of proteins and to the success of rational drug design and other biotechnology applications, obtaining high quality crystals is a major bottleneck to progress. The major means of obtaining crystals is by massive-scale screening of a target protein solution with numerous crystallizing agents. However, when crystals appear in these screens, one cannot easily know if they are crystals of protein, salt, or any other molecule that happens to be present in the trials. We present here a method based on Attenuated Total Reflection (ATR)-FT-IR imaging that reliably identifies protein crystals through a combination of chemical specificity and the visualizing capability of this approach, thus solving a major hurdle in protein crystallization. ATR-FT-IR imaging was successfully applied to study the crystallization of thaumatin and lysozyme in a high-throughput manner, simultaneously from six different solutions. This approach is fast as it studies protein crystallization in situ and provides an opportunity to examine many different samples under a range of conditions.

In the twelve years since styrene maleic acid (SMA) was first used to extract and purify a membrane protein within a native lipid bilayer, this technological breakthrough has provided insight into the structural and functional details of protein-lipid interactions. Most recently, advances in cryo-EM have demonstrated that SMA-extracted membrane proteins are a rich-source of structural data. For example, it has been possible to resolve the details of annular lipids and protein-protein interactions within complexes, the nature of lipids within central cavities and binding pockets, regions involved in stabilising multimers, details of terminal residues that would otherwise remain unresolved and the identification of physiologically relevant states. Functionally, SMA extraction has allowed the analysis of membrane proteins that are unstable in detergents, the characterization of an ultrafast component in the kinetics of electron transfer that was not possible in detergent-solubilised samples and quantitative, real-time measurement of binding assays with low concentrations of purified protein. While the use of SMA comes with limitations such as its sensitivity to low pH and divalent cations, its major advantage is maintenance of a protein's lipid bilayer. This has enabled researchers to view and assay proteins in an environment close to their native ones, leading to new structural and mechanistic insights. [ABSTRACT FROM AUTHOR]

Background Pichia pastoris is a widely-used host for recombinant protein production; expression is typically driven by methanol-inducible alcohol oxidase (AOX) promoters. Recently this system has become an important source of recombinant G protein-coupled receptors (GPCRs) for structural biology and drug discovery. The influence of diverse culture parameters (such as pH, dissolved oxygen concentration, medium composition, antifoam concentration and culture temperature) on productivity has been investigated for a wide range of recombinant proteins in P. pastoris. In contrast, the impact of the pre-induction phases on yield has not been as closely studied. In this study, we examined the pre-induction phases of P. pastoris bioreactor cultivations producing three different recombinant proteins: the GPCR, human A2a adenosine receptor (hA2aR), green fluorescent protein (GFP) and human calcitonin gene-related peptide receptor component protein (as a GFP fusion protein; hCGRP-RCP-GFP). Results Functional hA2aR was detected in the pre-induction phases of a 1 L bioreactor cultivation of glycerol-grown P. pastoris. In a separate experiment, a glycerol-grown P. pastoris strain secreted soluble GFP prior to methanol addition. When glucose, which has been shown to repress AOX expression, was the pre-induction carbon source, hA2aR and GFP were still produced in the pre-induction phases. Both hA2aR and GFP were also produced in methanolfree cultivations; functional protein yields were maintained or increased after depletion of the carbon source. Analysis of the pre-induction phases of 10 L pilot scale cultivations also demonstrated that pre-induction yields were at least maintained after methanol induction, even in the presence of cytotoxic concentrations of methanol. Additional bioreactor data for hCGRP-RCP-GFP and shake-flask data for GFP, horseradish peroxidase (HRP), the human tetraspanins hCD81 and CD82, and the tight-junction protein human claudin-1, demonstrated that bioreactor but not shake flask cultivations exhibit recombinant protein production in the pre-induction phases of P. pastoris cultures. Conclusions The production of recombinant hA2aR, GFP and hCGRP-RCP-GFP can be detected in bioreactor cultivations prior to methanol induction, while this is not the case for shake-flask cultivations of GFP, HRP, hCD81, hCD82 and human claudin-1. This confirms earlier suggestions of leaky expression from AOX promoters, which we report here for both glycerol- and glucose-grown cells in bioreactor cultivations. These findings suggest that the productivity of AOX-dependent bioprocesses is not solely dependent on induction by methanol. We conclude that in order to maximize total yields, pre-induction phase cultivation conditions should be optimized, and that increased specific productivity may result in decreased biomass yields. [ABSTRACT FROM AUTHOR]

The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-β-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1∶2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81. [ABSTRACT FROM AUTHOR]

The dipeptide carnosine (ß-alanyl-L-histidine) has contrasting but beneficial effects on cellular activity. It delays cellular senescence and rejuvenates cultured senescent mammalian cells. However, it also inhibits the growth of cultured tumour cells. Based on studies in several organisms, we speculate that carnosine exerts these apparently opposing actions by affecting energy metabolism and/or protein homeostasis (proteostasis). Specific effects on energy metabolism include the dipeptide's influence on cellular ATP concentrations. Carnosine's ability to reduce the formation of altered proteins (typically adducts of methylglyoxal) and enhance proteolysis of aberrant polypeptides is indicative of its influence on proteostasis. Furthermore these dual actions might provide a rationale for the use of carnosine in the treatment or prevention of diverse age-related conditions where energy metabolism or proteostasis are compromised. These include cancer, Alzheimer's disease, Parkinson's disease and the complications of type-2 diabetes (nephropathy, cataracts, stroke and pain), which might all benefit from knowledge of carnosine's mode of action on human cells. [ABSTRACT FROM AUTHOR]

Background: Recombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture. Yeast is a well-established recombinant host cell for these purposes. In this study we wanted to investigate whether our respiratory Saccharomyces cerevisiae strain, TM6*, could be used to enhance the productivity of recombinant proteins over that obtained from corresponding wild type, respiro-fermentative strains when cultured under the same laboratory conditions. Results: Here we demonstrate at least a doubling in productivity over wild-type strains for three recombinant membrane proteins and one recombinant soluble protein produced in TM6* cells. In all cases, this was attributed to the improved biomass properties of the strain. The yield profile across the growth curve was also more stable than in a wild-type strain, and was not further improved by lowering culture temperatures. This has the added benefit that improved yields can be attained rapidly at the yeast's optimal growth conditions. Importantly, improved productivity could not be reproduced in wild-type strains by culturing them under glucose fed-batch conditions: despite having achieved very similar biomass yields to those achieved by TM6* cultures, the total volumetric yields were not concomitantly increased. Furthermore, the productivity of TM6* was unaffected by growing cultures in the presence of ethanol. These findings support the unique properties of TM6* as a microbial cell factory. Conclusions: The accumulation of biomass in yeast cell factories is not necessarily correlated with a proportional increase in the functional yield of the recombinant protein being produced. The respiratory S. cerevisiae strain reported here is therefore a useful addition to the matrix of production hosts currently available as its improved biomass properties do lead to increased volumetric yields without the need to resort to complex control or cultivation schemes. This is anticipated to be of particular value in the production of challenging targets such as membrane proteins. [ABSTRACT FROM AUTHOR]

The Saccharomyces cerevisiae gene FPS1 encodes an aquaglyceroporin of the major intrinsic protein (MIP) family. The main function of Fps1p seems to be the efflux of glycerol in the adaptation of the yeast cell to lower external osmolarity. Fps1p is an atypical member of the family, because the protein is much larger (669 amino acids) than most MIPs due to long hydrophilic extensions in both termini. We have shown previously that a short domain in the N-terminal extension of the protein is required for restricting glycerol transport through the channel (Tamás, M. J., Karlgren, S., Bill, R. M., Hedfalk, K., Allegri, L., Ferreira, M., Thevelein, J. M., Rydström, J., Mullins, J. G. L., and Hohmann, S. (2003) J. Biol. Chem. 278, 6337-6345). Deletion of the N-terminal domain results in an unregulated channel, loss of glycerol, and osmosensitivity. In this work we have investigated the role of the Fps1p C terminus (139 amino acids). A set of eight truncations has been constructed and tested in vivo in a yeast fps1Δ strain. We have performed growth tests, membrane localization following cell fractionation, and glycerol accumulation measurements as well as an investigation of the osmotic stress response. Our results show that the C-terminal extension is also involved in restricting transport through Fps1p. We have identified a sequence of 12 amino acids, residues 535-546, close to the sixth transmembrane domain. This element seems to be important for controlling Fps1p function. Similar to the N-terminal domain, the C-terminal domain is amphiphilic and has a potential to dip into the membrane. [ABSTRACT FROM AUTHOR]

In recent years, the scientific community has begun to realise that the structure–function analysis of membrane proteins has lagged considerably behind that of their soluble counterparts. A boom in the field of membrane protein biology has resulted in the tailoring of techniques for the cloning, expression, purification and characterisation of these somewhat intractable proteins and most notably in the optimisation of several alternative host systems for this purpose. This Review Article summarises the use of yeast as a host. Compared with other hosts, it is clear that yeast combines the advantages of eukaryotes with the ease of handling of prokaryotes. Moreover, this organism provides membrane protein biologists with a panacea for structure–function analyses, not least because the tools of yeast genetics are at their disposal. [ABSTRACT FROM AUTHOR]