Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris

<p>Abstract</p> <p>Background</p> <p>When performing functional and structural studies, large quantities of pure protein are desired. Most membrane proteins are however not abundantly expressed in their native tissues, which in general rules out purification from natural sources. Heterologous expression, especially of eukaryotic membrane proteins, has also proven to be challenging. The development of expression systems in insect cells and yeasts has resulted in an increase in successful overexpression of eukaryotic proteins. High yields of membrane protein from such hosts are however not guaranteed and several, to a large extent unexplored, factors may influence recombinant expression levels. In this report we have used four isoforms of aquaporins to systematically investigate parameters that may affect protein yield when overexpressing membrane proteins in the yeast <it>Pichia pastoris</it>.</p> <p>Results</p> <p>By comparing clones carrying a single gene copy, we show a remarkable variation in recombinant protein expression between isoforms and that the poor expression observed for one of the isoforms could only in part be explained by reduced transcript levels. Furthermore, we show that heterologous expression levels of all four aquaporin isoforms strongly respond to an increase in recombinant gene dosage, independent of the amount of protein expressed from a single gene copy. We also demonstrate that the increased expression does not appear to compromise the protein folding and the membrane localisation.</p> <p>Conclusions</p> <p>We report a convenient and robust method based on qPCR to determine recombinant gene dosage. The method is generic for all constructs based on the pPICZ vectors and offers an inexpensive, quick and reliable means of characterising recombinant <it>P. pastoris </it>clones. By using this method we show that: (1) heterologous expression of all aquaporins investigated respond strongly to an increase in recombinant gene dosage (2) expression from a single recombinant gene copy varies in an isoform dependent manner (3) the poor expression observed for AtSIP1;1 is mainly caused by posttranscriptional limitations. The protein folding and membrane localisation seems to be unaffected by increased expression levels. Thus a screen for elevated gene dosage can routinely be performed for identification of <it>P. pastoris </it>clones with high expression levels of aquaporins and other classes of membrane proteins.</p

Well-being and mental health in later life: The development and validation of the well-being evaluation scale.

Wellbeing is a complex construct and to date there has been great inconsistency across measures of wellbeing with respect to their theoretical foundations, dimensions, composition, and psychometric properties. The need for wellbeing measures that have robust epistemological and ontological frameworks and sound psychometric properties is evident as a basis for effective care planning, outcome evaluation and research. The following paper describes the development and psychometric evaluation of the Wellbeing Evaluation Scale (WES), long and short forms, from an analysis of data drawn from a representative population of 203 adults aged 55 years and over. Following an initial pilot study (n = 98), a repeated investigation involved a representative sample of 203 adults over the age of 55 who were recruited from the West Midlands for this study. The findings suggest that the WES is a reliable, valid, and representative measure of wellbeing in adults over 55. It is concluded that the WES can be considered as a reliable tool for the purposes of assessment, care planning and research. </jats:p