Overexpression of membrane proteins is often essential for structural and functional studies, but yields are frequently too low. An understanding of the physiological response to overexpression is needed to improve such yields. Therefore, we analyzed the consequences of overexpression of three different membrane proteins (YidC, YedZ, and LepI) fused to green fluorescent protein (GFP) in the bacterium Escherichia coli and compared this with overexpression of a soluble protein, GST-GFP. Proteomes of total lysates, purified aggregates, and cytoplasmic membranes were analyzed by one-and two-dimensional gel electrophoresis and mass spectrometry complemented with flow cytometry, microscopy, Western blotting, and pulse labeling experiments. Composition and accumulation levels of protein complexes in the cytoplasmic membrane were analyzed with improved two-dimensional blue native PAGE. Overexpression of the three membrane proteins, but not soluble GST-GFP, resulted in accumulation of cytoplasmic aggregates containing the overexpressed proteins, chaperones (DnaK/J and GroEL/ S), and soluble proteases (HslUV and ClpXP) as well as many precursors of periplasmic and outer membrane proteins. This was consistent with lowered accumulation levels of secreted proteins in the three membrane protein overexpressors and is likely to be a direct consequence of saturation of the cytoplasmic membrane protein translocation machinery. Importantly accumulation levels of respiratory chain complexes in the cytoplasmic membrane were strongly reduced. Induction of the acetate-phosphotransacetylase pathway for ATP production and a downregulated tricarboxylic acid cycle indicated the activation of the Arc two-component system, which mediates adaptive responses to changing respiratory states. This study provides a basis for designing rational strategies to improve yields of membrane protein overexpression in E. coli.

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MICA and MICB are ligands of the NKG2D receptor and thereby influence NK and T cell activity. MICA/B gene polymorphisms, expression levels and the amount of soluble MICA/B in the serum have been linked to autoimmune diseases, infections, and cancer. In hematopoietic stem cell transplantation, MICA matching between donor and patient has been correlated with reduced acute and chronic graft-vs.-host disease and improved survival. Hence, we developed an extremely cost-efficient high-throughput workflow for genotyping MICA/B for newly registered potential stem cell donors. Since mid-2017, we have genotyped over two million samples using NGS amplicon sequencing for MICA/B exons 2-5. In donors of German origin, MICA * 008 is the most common MICA allele with a frequency of 42.3%. It is followed by MICA * 002 (11.7%) and MICA * 009 (8.8%). The three most common MICB alleles are MICB * 005 (43.9%), MICB * 004 (21.7%), and MICB * 002 (18.9%). In general, MICB is less diverse than MICA and only 6 alleles, instead of 15, account for a cumulative allele frequency of 99.5%. In 0.5% of the samples we observed at least one allele of MICA or MICB which has so far not been reported to the IPD/IMGT-HLA database. By providing MICA/B typed voluntary donors, clinicians become empowered to include MICA/B into their donor selection process to further improve unrelated hematopoietic stem cell transplantation.

Currently, stem cell donor registries include more than 35 million potential donors worldwide to provide HLA‐matched stem cell products for patients in need of an unrelated donor transplant. DKMS is a leading stem cell donor registry with more than 9 million donors from Germany, Poland, the United States, the United Kingdom, India and Chile. DKMS donors have donated hematopoietic stem cells more than 80,000 times. Many aspects of donor registry work are closely related to topics from immunogenetics or population genetics. In this two‐part review article, we describe, analyse and discuss these areas of donor registry work by using the example of DKMS. Part 1 of the review gives a general overview on DKMS and includes typical donor registry activities with special focus on the HLA system: high‐throughput HLA typing of potential stem cell donors, HLA haplotype frequencies and resulting matching probabilities, and donor file optimization with regard to HLA diversity.

Phosphorylation of the Marburg virus nucleoprotein NP is distributed over 7 regions (I-VII) in its C-terminus. The exact localization of phosphorylated amino acids and function of NP phosphorylation are unknown. Here, we show that the major phosphate acceptor sites in NP region II are serine 446 and serines 453-455; the latter are located in a cluster of 6 serine residues (aa 450-455). The function of phosphorylation in region II was tested using an infectious virus-like particle assay. Phosphorylation influenced reporter gene activity that reflects viral transcription and replication. An NP mutant mimicking 3 phosphorylated serine residues at position 453-455 supported reporter gene activity better than wild-type NP. Negative charges at positions 450-452 and when the serine cluster was completely substituted by alanine inhibited reporter gene activity significantly. These data support the idea that phosphorylation of NP region II modulates viral RNA synthesis in transcription and/or replication.