Correlation of cell growth and heterologous protein production by Saccharomyces cerevisiae

With the increasing demand for biopharmaceutical proteins and industrial enzymes, it is necessary to optimize the production by microbial fermentation or cell cultures. Yeasts are well established for the production of a wide range of recombinant proteins, but there are also some limitations; e.g., metabolic and cellular stresses have a strong impact on recombinant protein production. In this work, we investigated the effect of the specific growth rate on the production of two different recombinant proteins. Our results show that human insulin precursor is produced in a growth-associated manner, whereas alpha-amylase tends to have a higher yield on substrate at low specific growth rates. Based on transcriptional analysis, we found that the difference in the production of the two proteins as function of the specific growth rate is mainly due to differences in endoplasmic reticulum processing, protein turnover, cell cycle, and global stress response. We also found that there is a shift at a specific growth rate of 0.1 h(-1) that influences protein production. Thus, for lower specific growth rates, the alpha-amylase and insulin precursor-producing strains present similar cell responses and phenotypes, whereas for higher specific growth rates, the two strains respond differently to changes in the specific growth rate

Neurosyphilis manifesting with unilateral visual loss and hyponatremia: a case report

<p>Abstract</p> <p>Background</p> <p>Syphilis is called the chameleon of the diseases due to its variety of its clinical presentations, potentially affecting every organ of the body. Incidence of this ancient disease is once again on the increase worldwide.</p> <p>Case presentation</p> <p>We here report an unusual case of neurosyphilis manifesting with unilateral visual loss and hyponatremia. The patient also had primary syphilitic lesions and was concomitantly diagnosed with Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) infection. Treatment with ceftriaxone and prednisolone, completely resolved the hyponatremia and visual acuity was partially restored.</p> <p>Conclusion</p> <p>Awareness of syphilis as a differential diagnosis is important as previously unreported presentations of neurosyphilis can arise, especially in HIV infected patients.</p

Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

International audienceBACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein

Functional expression of plant membrane proteins in Lactococcus lactis.

International audienceThe study of most membrane proteins remains challenging due to their hydrophobicity and their low natural abundance in cells. Lactococcus lactis, a Gram-positive lactic bacterium, has been traditionally used in food fermentations and is nowadays widely used in biotechnology for large-scale production of heterologous proteins. This system has been successfully used for the production of prokaryotic and eukaryotic membrane proteins. The purpose of this chapter is to provide detailed protocols for (1) the expression of plant peripheral or intrinsic membrane proteins and then for (2) their solubilization, from Lactococcus membranes, for further purification steps and biochemical characterization

Drug Resistance Is Conferred on the Model Yeast Saccharomyces cerevisiae

[Image: see text] ABCB5, an ATP-binding cassette (ABC) transporter, is highly expressed in melanoma cells, and may contribute to the extreme resistance of melanomas to chemotherapy by efflux of anti-cancer drugs. Our goal was to determine whether we could functionally express human ABCB5 in the model yeast Saccharomyces cerevisiae, in order to demonstrate an efflux function for ABCB5 in the absence of background pump activity from other human transporters. Heterologous expression would also facilitate drug discovery for this important target. DNAs encoding ABCB5 sequences were cloned into the chromosomal PDR5 locus of a S. cerevisiae strain in which seven endogenous ABC transporters have been deleted. Protein expression in the yeast cells was monitored by immunodetection using both a specific anti-ABCB5 antibody and a cross-reactive anti-ABCB1 antibody. ABCB5 function in recombinant yeast cells was measured by determining whether the cells possessed increased resistance to known pump substrates, compared to the host yeast strain, in assays of yeast growth. Three ABCB5 constructs were made in yeast. One was derived from the ABCB5-β mRNA, which is highly expressed in human tissues but is a truncation of a canonical full-size ABC transporter. Two constructs contained full-length ABCB5 sequences: either a native sequence from cDNA or a synthetic sequence codon-harmonized for S. cerevisiae. Expression of all three constructs in yeast was confirmed by immunodetection. Expression of the codon-harmonized full-length ABCB5 DNA conferred increased resistance, relative to the host yeast strain, to the putative substrates rhodamine 123, daunorubicin, tetramethylrhodamine, FK506, or clorgyline. We conclude that full-length ABCB5 can be functionally expressed in S. cerevisiae and confers drug resistance

Structure and mechanism of the ECF-type ABC transporter for thiamin

Guus Erkens’ PhD thesis is about the thiamin (vitamin B1) transport protein ThiT from the bacterium Lactococcus lactis. During his research project he has investigated the recognition of thiamin by ThiT. Also he has determined the three-dimensional structure of ThiT using X-ray crystallography. These results may provide a starting point for the development of antibiotics. The uptake of vitamins requires specific transport proteins residing in the cell membrane. Studying membrane proteins is notoriously difficult. Our knowledge of these protein mechanisms is therefore limited. Alike humans and other organisms bacteria depend on vitamins for their survival. Many bacteria are capable of producing vitamins but often depend on the uptake of vitamins from their environment as well. The determination of the three-dimensional structure of a membrane protein is technically very challenging. Therefore very few membrane protein structures are available. The results presented in this thesis have yielded great insight in the mechanism of ThiT and in vitamin transport in general. Proteins are constructed of long chains folded in a complex three-dimensional pattern. Their function is largely determined by this pattern. In co-operation with other researchers more vitamin transport proteins similar to ThiT have been identified. These findings have lead to a more detailed biochemical study of ThiT. After purifying the protein from Lactococcus lactis cell membranes, the interaction of ThiT with thiamin could be investigated. Thiamin binding to ThiT turned out to be of a very high affinity. Given these results Guus Erkens concluded that high affinity binding allows bacteria to take up thiamin when it is available in very low concentrations.