Tuning microbial hosts for membrane protein production

The last four years have brought exciting progress in membrane protein research. Finally those many efforts that have been put into expression of eukaryotic membrane proteins are coming to fruition and enable to solve an ever-growing number of high resolution structures. In the past, many skilful optimization steps were required to achieve sufficient expression of functional membrane proteins. Optimization was performed individually for every membrane protein, but provided insight about commonly encountered bottlenecks and, more importantly, general guidelines how to alleviate cellular limitations during microbial membrane protein expression. Lately, system-wide analyses are emerging as powerful means to decipher cellular bottlenecks during heterologous protein production and their use in microbial membrane protein expression has grown in popularity during the past months

False Positive Immunoglobulin M Antibody to Cytomegalovirus in Child with Infectious Mononucleosis Caused by Epstein-Barr Virus Infection

A 16-month-old boy was admitted because of cough that had lasted for 10 days. The patient showed severe hepatomegaly incidentally, and dual positivity of Immunoglobulin (Ig) M to Epstein-Barr virus (EBV) viral capsid antigen (VCA) and cytomegalovirus (CMV). On the basis of seroconversion to Epstein-Barr nuclear antigen (EBNA) Ig G positivity and reduced CMV Ig M titer with persistently negative CMV Ig G, a definite diagnosis of EBV-induced infectious mononucleosis was established 1 year 2 month later

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

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

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

Panretinale photokoagulation mit dem mikrogepulsten diodenlaser bei proliferativer diabetischer retinopathie

Repetitive, short-pulse laser can coagulate the retinal pigment epithelium selectively, while sparing the adjacent outer neuroretina and the choroid. Therefore the functional defects caused by panretinal photocoagulation with conventional laser delivery systems may be reduced or even avoided using a micropulsed diode laser. A panretinal laser treatment was performed in 6 patients with proliferative diabetic retinopathy using an argon laser on one and a micropulsed diode laser on the fellow eye. Pre- and posttreatment examination consisted besides of a complete ophthalmological status, a fluorescein angiography, visual fields and of electrophysiological tests. The argon laser eyes showed a profound loss of response in the scotopic ERG and more absolut scotomas in the peripheral visual fields compared to the micropulsed eyes. Most of the laser burns caused by the micropulsed diode laser could not be detected neither ophthalmoscopically nor angiographically. Some lesions were visible as small laser scars. No regression of the proliferations could be achieved with the diode laser after a follow-up of at least 5 months. The functional damage after panretinal photocoagulation can be reduced using a micropulsed diode laser, but no stabilisation of the proliferative retinopathy could be obtained at the energy levels used.</p