Sue Macauley-Patrick

Bio: Sue Macauley-Patrick is an academic researcher from University of Strathclyde. The author has contributed to research in topics: Sorbose & Heterologous expression. The author has an hindex of 3, co-authored 3 publications receiving 1194 citations.

Heterologous protein production using the Pichia pastoris expression system.

Abstract: 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.

Attenuated total reflectance Fourier transform mid-infrared spectroscopic quantification of sorbitol and sorbose during a Gluconobacter biotransformation process.

Abstract: Mid-infrared spectroscopy (MIRS) was used to simultaneously detect and predict concentrations of D-sorbitol and L-sorbose during a Gluconobacter suboxydans biotransformation Quantitative models for both these compounds were developed for the entire time-course of the process and validated externally using samples not included in the original modelling exercise, giving standard errors of prediction of 329 and 33% for sorbitol and sorbose, respectively, and a correlation coefficient close to 1

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production.

Abstract: Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. pastoris to such high titres. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation. The last few years have been particularly fruitful because of numerous activities in improving the expression of such complex proteins with a focus on either protein engineering or on engineering the protein expression host P. pastoris. This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains. Breakthroughs in membrane protein expression are discussed alongside numerous commercial applications of P. pastoris derived proteins.

Efflux-Mediated Antifungal Drug Resistance

Abstract: Fungi cause serious infections in the immunocompromised and debilitated, and the incidence of invasive mycoses has increased significantly over the last 3 decades. Slow diagnosis and the relatively few classes of antifungal drugs result in high attributable mortality for systemic fungal infections. Azole antifungals are commonly used for fungal infections, but azole resistance can be a problem for some patient groups. High-level, clinically significant azole resistance usually involves overexpression of plasma membrane efflux pumps belonging to the ATP-binding cassette (ABC) or the major facilitator superfamily class of transporters. The heterologous expression of efflux pumps in model systems, such Saccharomyces cerevisiae, has enabled the functional analysis of efflux pumps from a variety of fungi. Phylogenetic analysis of the ABC pleiotropic drug resistance family has provided a new view of the evolution of this important class of efflux pumps. There are several ways in which the clinical significance of efflux-mediated antifungal drug resistance can be mitigated. Alternative antifungal drugs, such as the echinocandins, that are not efflux pump substrates provide one option. Potential therapeutic approaches that could overcome azole resistance include targeting efflux pump transcriptional regulators and fungal stress response pathways, blockade of energy supply, and direct inhibition of efflux pumps.

Recombinant Protein Production in Yeasts

Abstract: Recombinant protein production is a multibillion-dollar market. The development of a new product begins with the choice of a production host. While one single perfect host for every protein does not exist, several expression systems ranging from bacterial hosts to mammalian cells have been established. Among them, yeast cell factories combine the advantages of being single cells, such as fast growth and easy genetic manipulation, as well as eukaryotic features including a secretory pathway leading to correct protein processing and post-translational modifications. In this respect, especially the engineering of yeast glycosylation to produce glycoproteins of human-like glycan structures is of great interest. Additionally, different attempts of cellular engineering as well as the design of different production processes that are leading to improved productivities are presented. With the advent of cheaper next-generation sequencing techniques, systems biotechnology approaches focusing on genome scale analyses will advance and accelerate yeast cell factories and thus recombinant protein production processes in the near future. In this review we summarize advantages and limitations of the main and most promising yeast hosts, including Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha as those presently used in large scale production of heterologous proteins.