RESUMEN
Modular Cloning (MoClo) is based on libraries of standardized genetic parts that can be directionally assembled via Golden Gate cloning in one-pot reactions into transcription units and multigene constructs. Here, a team of bachelor students established a MoClo toolkit for the protist Leishmania tarentolae in the frame of the international Genetically Engineered Machine (iGEM) competition. Our modular toolkit is based on a domesticated version of a commercial LEXSY expression vector and comprises 34 genetic parts encoding various affinity tags, targeting signals as well as fluorescent and luminescent proteins. We demonstrated the utility of our kit by the successful production of 16 different tagged versions of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein in L. tarentolae liquid cultures. While highest yields of secreted recombinant RBD were obtained for GST-tagged fusion proteins 48 h post induction, C-terminal peptide tags were often degraded and resulted in lower yields of secreted RBD. Fusing secreted RBD to a synthetic O-glycosylation SP20 module resulted in an apparent molecular mass shift around 10 kDa. No disadvantage regarding the production of RBD was detected when the three antibiotics of the LEXSY system were omitted during the 48-h induction phase. Furthermore, the successful purification of secreted RBD from the supernatant of L. tarentolae liquid cultures was demonstrated in pilot experiments. In summary, we established a MoClo toolkit and exemplified its application for the production of recombinant proteins in L. tarentolae.
RESUMEN
The spike protein is the major protein on the surface of coronaviruses. It is therefore the prominent target of neutralizing antibodies and consequently the antigen of all currently admitted vaccines against SARS-CoV-2. Since it is a 1,273-amino acids glycoprotein with 22 N-linked glycans, the production of functional, full-length spike protein was limited to higher eukaryotes. Here we report the production of full-length SARS-CoV-2 spike protein - lacking the C-terminal membrane anchor - as a secreted protein in the prefusion-stabilized conformation in the unicellular green alga Chlamydomonas reinhardtii. We show that the spike protein is efficiently cleaved at the furin cleavage site during synthesis in the alga and that cleavage is abolished upon mutation of the multi-basic cleavage site. We could enrich the spike protein from culture medium by ammonium sulfate precipitation and demonstrate its functionality based on its interaction with recombinant ACE2 and ACE2 expressed on human 293T cells. Chlamydomonas reinhardtii is a GRAS organism that can be cultivated at low cost in simple media at a large scale, making it an attractive production platform for recombinant spike protein and other biopharmaceuticals in low-income countries.
RESUMEN
Germany is facing a shortage of young family doctors. In search of possible reasons the aim of this study was to explore the perception of specialists on family doctors. Within a qualitative study 16 medical specialists from different fields in hospital and outpatient care setting were interviewed. Interviews were analysed using qualitative content analysis according to Mayring. Most of the interviewed specialists have a positive view on family doctors although a certain depreciative assumption is resonated in a number of statements. According to the specialists, family doctors enjoy a high status in public, even if social processes of change may have a negative influence on their rather old-fashioned image. Specialists find that family medicine is underrepresented in university education suffering from an upgrading of specialized disciplines. Altogether the majority of the interviewed specialists certify family doctors in Germany a positive image. Lecturer in medical education and training should be aware of their key role in the career choices of young trainees and avoid degradation or upgrading of certain medical disciplines. Interlinked measures on different levels focusing on the improvement of working conditions and representation at the universities would be needed to regain attractiveness for the family doctor's profession as a career choice for young doctors.
RESUMEN
BACKGROUND: Promoting satiety and repressing appetite is one major goal in the dietetic therapy of obesity. In the past, several studies investigated the effect of different macronutrients, especially protein and carbohydrates, on short- and long-term satiety in humans. This paper aims to directly compare the effect of protein, rolled oats (low glycemic index), sugar or cornflakes (high glycemic index), and walnuts (high amount of omega-3 fatty acids) as ingredients of a yoghurt breakfast on short-term hunger and satiety in one setting. A second objective was to study the effect of these yoghurt breakfasts on mental state. METHODS: 14 healthy male volunteers participated in this randomized, controlled, cross-over design study. After consuming the different test meals, volunteers repeatedly completed 2 questionnaires over a total of 3 h. RESULTS: The protein meal showed the highest satiety scores and the controls (low-calorie yoghurt) the lowest. The other test meals were not different among each other. Regarding mental state (mood, fatigue, and calmness), no significant difference between the test meals and the low-calorie control was observed. CONCLUSION: The glycemic index does not seem to modify satiety in this short-term setting. The similar mental state between low- and high-calorie breakfasts deserves further investigations.
