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Using the methods developed to study protein DNA complexes, a small number of workers has begun to characterize the interactions of lupus anti-DNA antibodies with their DNA antigens. These studies indicate that anti-ssDNA generally possess a high affinity for poly(dT) and use aromatic side-chains on complex formation, in common with many previously reported anti-ssDNA, and several DNA-binding proteins. We find that our anti-ssDNA differ from these latter two species in that they are not cross-reactive with non-nucleic acid antigens, unlike many other anti-ssDNA, and their complexation does not appear to be accompanied by significant cation release, as for some DNA-binding proteins. Moreover, in some cases our anti-ssDNA are apparently sequence-specific. Due to the small number of anti-dsDNA mAbs studied, our current understanding of these proteins is limited. However, we have recently obtained a high-resolution crystal structure of 4B2, that reacts with both ss- and dsDNA. This structure should enable us to facilitate mutagenesis experiments to identify the molecular features of dsDNA recognition. One can envisage several practical benefits of applying a systematic biophysical approach to studying lupus anti-DNA·DNA. For example, once the molecular basis of anti-DNA·DNA interactions has been defined, it may be possible to use anti-DNA as biochemical reagents such as repressors of protein-DNA binding or as catalysts that manipulate DNA. Indeed, the enormous diversity of the immune repertoire and the case with which anti-DNA can be customized by site-directed and random mutagenesis provides the potential to generate anti-DNA with a wide range of properties. Alternatively, by understanding the molecular basis of anti-DNA·DNA interactions it may be possible to identify molecules that disrupt the specific interaction between anti-DNA and their DNA antigens. Such molecules could eventually form the basis of new agents to treat immune-complex mediated kidney damage and have fewer complications than the nonspecific agents currently used to treat lupus. In fact, by screening combinatorial small molecule libraries, we have already identified several promising anti-DNA antagonists.58 Thus, we believe that the next few years will be an exciting period in anti-DNA research. We review some common approaches that have been used to examine the specificity, affinity, and mode of binding of lupus anti-DNA for DNA antigens, We highlight the recent use of biophysical methods that have been used to study DNA-binding proteins, such as transcription factors, and demonstrate their utility when used in the study of lupus anti-DNA. ga]473|The application of set theory to combinatorial processes provides valuable tools for the planning, description, execution, and evaluation of combinatorial events.

Background The production of high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences that has yet to be addressed in a truly rational manner. Typically eukaryotic protein production experiments have relied on varying expression construct cassettes such as promoters and tags, or culture process parameters such as pH, temperature and aeration to enhance yields. These approaches require repeated rounds of trial-and-error optimization and cannot provide a mechanistic insight into the biology of recombinant protein production. We published an early transcriptome analysis that identified genes implicated in successful membrane protein production experiments in yeast. While there has been a subsequent explosion in such analyses in a range of production organisms, no one has yet exploited the genes identified. The aim of this study was to use the results of our previous comparative transcriptome analysis to engineer improved yeast strains and thereby gain an understanding of the mechanisms involved in high-yielding protein production hosts. Results We show that tuning BMS1 transcript levels in a doxycycline-dependent manner resulted in optimized yields of functional membrane and soluble protein targets. Online flow microcalorimetry demonstrated that there had been a substantial metabolic change to cells cultured under high-yielding conditions, and in particular that high yielding cells were more metabolically efficient. Polysome profiling showed that the key molecular event contributing to this metabolically efficient, high-yielding phenotype is a perturbation of the ratio of 60S to 40S ribosomal subunits from approximately 1:1 to 2:1, and correspondingly of 25S:18S ratios from 2:1 to 3:1. This result is consistent with the role of the gene product of BMS1 in ribosome biogenesis. Conclusion This work demonstrates the power of a rational approach to recombinant protein production by using the results of transcriptome analysis to engineer improved strains, thereby revealing the underlying biological events involved.

Background Optimisation of culture conditions for the expression and production of important therapeutic biologics such as recombinant proteins, antibody-fragments and fusion proteins is a key element in the rapid and cost effective manufacture of these important molecules [1]. The factors to be considered when producing proteins from microorganisms such as Saccharomyces cerevisiae or Pichia pastoris include: pH, temperature, carbon and nitrogen sources and the essential oxygen requirement. The demand for oxygen by a microorganism can be met by aerating the medium that it is growing in, which is most often done by sparging sterile air through the medium.

Background: Emerging evidence supports the view that (AQP) aquaporin water channels are regulators of transcellular\ud water flow. Consistentwith their expression in most tissues, AQPs are associatedwith diverse physiological\ud and pathophysiological processes.\ud Scope of review: AQP knockout studies suggest that the regulatory role of AQPs, rather than their action as passive\ud channels, is their critical function. Transport through all AQPs occurs by a common passive mechanism, but their\ud regulation and cellular distribution varies significantly depending on cell and tissue type; the role of AQPs in cell\ud volumeregulation (CVR) is particularly notable. This reviewexamines the regulatory role of AQPs in transcellular\ud water flow, especially in CVR.We focus on key systems of the human body, encompassing processes as diverse as\ud urine concentration in the kidney to clearance of brain oedema.\ud Major conclusions: AQPs are crucial for the regulation of water homeostasis, providing selective pores for the\ud rapidmovement ofwater across diverse cellmembranes and playing regulatory roles in CVR. Gatingmechanisms\ud have been proposed for human AQPs, but have only been reported for plant andmicrobial AQPs. Consequently, it\ud is likely that the distribution and abundance of AQPs in a particular membrane is the determinant of membrane\ud water permeability and a regulator of transcellular water flow.\ud General significance: Elucidating the mechanisms that regulate transcellular water flow will improve our understanding\ud of the human body in health and disease. The central role of specific AQPs in regulating water homeostasis\ud will provide routes to a range of novel therapies. This article is part of a Special Issue entitled Aquaporins.

The production of membrane proteins of high purity and in satisfactory yields is crucial for biomedical research. Due to their involvement in various cellular processes, membrane proteins have increasingly become some of the most important drug targets in modern times. Therefore, their structural and functional characterization is a high priority. However, protein expression has always been more challenging for membrane proteins than for soluble proteins. In this review, we present four of the most commonly-used expression systems for eukaryotic membrane proteins. We describe the benefits and drawbacks of bacterial, yeast, insect and mammalian cells. In addition, we describe the different features (growth rate, yield, post-translational modifications) of each expression system, and how they are influenced by the construct design and modifications of the target gene. Cost-effective and fast-growing E. coli is mostly selected for the production of small, simple membrane proteins that, if possible, do not require post-translational modifications but has the potential for the production of bigger proteins as well. Yeast hosts are advantageous for larger and more complex proteins but for the most complex ones, insect or mammalian cells are used as they are the only hosts able to perform all the post-translational modifications found in human cells. A combination of rational construct design and host cell choice can dramatically improve membrane protein production processes.

Background We previously described the first respiratory Saccharomyces cerevisiae strain, KOY.TM6*P, by integrating the gene encoding a chimeric hexose transporter, Tm6*, into the genome of an hxt null yeast. Subsequently we transferred this respiratory phenotype in the presence of up to 50 g/L glucose to a yeast strain, V5 hxt1-7 Δ, in which only HXT1-7 had been deleted. In this study, we compared the transcriptome of the resultant strain, V5.TM6*P, with that of its wild-type parent, V5, at different glucose concentrations. Results cDNA array analyses revealed that alterations in gene expression that occur when transitioning from a respiro-fermentative (V5) to a respiratory (V5.TM6*P) strain, are very similar to those in cells undergoing a diauxic shift. We also undertook an analysis of transcription factor binding sites in our dataset by examining previously-published biological data for Hap4 (in complex with Hap2, 3, 5), Cat8 and Mig1, and used this in combination with verified binding consensus sequences to identify genes likely to be regulated by one or more of these. Of the induced genes in our dataset, 77% had binding sites for the Hap complex, with 72% having at least two. In addition, 13% were found to have a binding site for Cat8 and 21% had a binding site for Mig1. Unexpectedly, both the up- and down-regulation of many of the genes in our dataset had a clear glucose dependence in the parent V5 strain that was not present in V5.TM6*P. This indicates that the relief of glucose repression is already operable at much higher glucose concentrations than is widely accepted and suggests that glucose sensing might occur inside the cell. Conclusion Our dataset gives a remarkably complete view of the involvement of genes in the TCA cycle, glyoxylate cycle and respiratory chain in the expression of the phenotype of V5.TM6*P. Furthermore, 88% of the transcriptional response of the induced genes in our dataset can be related to the potential activities of just three proteins: Hap4, Cat8 and Mig1. Overall, our data support genetic remodelling in V5.TM6*P consistent with a respiratory metabolism which is insensitive to external glucose concentrations.