Recombinant production and purification of short hydrophobic Elastin-like polypeptides with low transition temperatures

Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. We report herein the recombinant expression of three hydrophobic ELP5 (VPGIG)(n) with variable lengths (n = 20, 40, 60) and sub-ambient transition temperatures. These ELPs were purified from the cytoplasmic soluble fraction of Escherichia coli by inverse transition cycling, and their exact molecular weight was confirmed by various mass spectrometry techniques. Transition temperatures of ELP20, ELP40, and ELP60 were measured at 18.6 degrees C, 12.4 degrees C and 11.7 degrees C, respectively

Exosomal sorting of the cytoplasmic domain of bovine leukemia virus TM Env protein

International audienceExosomes are small membrane vesicles that are released into the extracellular compartment as a consequence of fusion of multivesicular endosomes with the plasma membrane. To unravel the molecular basis of protein sorting into exosomes, we have made a chimeric protein containing the cytosolic domain of the transmembrane subunit of the viral Env protein of BLV and the ectodomain of CD8 (CDTM-BLV-CD8). When expressed in K562 cells known to constitutively secrete exosomes, the chimera was found to be very efficiently targeted to the released vesicles. Very interestingly, the cytosolic domain of the Env protein contains peptide motifs potentially recognized by components of the ESCRT machinery that could be related to chimera sorting into the vesicles. Then, quantifying the chimera secretion, we investigated the site of exosome biogenesis in K562 cells using a pharmacological approach. We present different arguments indicating that CDTM-BLV-CD8-containing exosomes are likely formed from a recycling endosomal/TGN compartment

Expression and purification of short hydrophobic elastin-like polypeptides with maltose-binding protein as a solubility tag

Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. The recombinant expression of hydrophobic elastin-like polypeptides is often difficult because they possess low transition temperatures, and therefore form aggregates at sub-ambient temperatures. To circumvent this difficulty, we expressed in Escherichia coli three hydrophobic ELPs (VPGIG)(n) with variable lengths (n = 20, 40, and 60) in fusion with the maltose-binding protein (MBP). Fusion proteins were soluble and yields of purified MBP-ELP ranged between 66 and 127 mg/L culture. After digestion of the fusion proteins by enterokinase, the ELF moiety was purified by using inverse transition cycling. The purified fraction containing ELP40 was slightly contaminated by traces of undigested fusion protein. Purification of ELP60 was impaired because of co-purification of the MBP tag during inverse transition cycling. ELP20 was successfully purified to homogeneity, as assessed by gel electrophoresis and mass spectrometry analyses. The transition temperature of ELP20 was measured at 15.4 degrees C in low salt buffer. In conclusion, this method can be used to produce hydrophobic ELF of low molecular mas

Use of the human hepcidin gene to build a positive-selection vector for periplasmic expression in Escherichia coli

Recombinant proteins are often produced in the periplasm of Escherichia coli because this facilitates the purification process. The oxidizing environment favors the formation of disulfide bridges. We showed that the periplasmic expression of the human hormone hepcidin 25 (Hep25) fused to the maltose binding protein (MBP) resulted in cell death. This toxicity was not observed when MBP-Hep25 accumulated in the bacterial cytoplasm, or when Hep25 was addressed to the periplasm without the MBP tag. We then modified the periplasmic expression vector pMALp2E to create pMALp2EH, a positive-selection vector with Hep25 as counterselection gene. (C) 2016 Elsevier Inc. All rights reserved

1 H, 13 C, 15 N NMR Resonance Assignments and Secondary Structure Determination of the Extra-Cellular Domain from the Human Proapoptotic TRAIL-R2 Death Receptor 5 (DR5-ECD)

International audienc

Production and purification of recombinant human hepcidin-25 with authentic N and C-termini

Hepcidin was first identified as an antimicrobial peptide present in human serum and urine. It was later demonstrated that hepcidin is the long-sought hormone that regulates iron homeostasis in mammals. Recombinant human Hepcidin-25 (Hepc25) was expressed in Pichia pastoris using a modified version of the pPICZ alpha A vector. Hepc25 was then purified by a simple two-step chromatographic process to obtain 1.9 mg of soluble recombinant human Hepc25 per liter of culture at 96% purity. The sequence of Hepc25 and the presence of four disulfide bridges were confirmed by mass spectrometry analyses, and the recombinant Hepc25 exhibited antibacterial activity. This protocol of production and purification is the first step toward the production of human Hepc25 at a greater scale. (C) 2015 Elsevier B.V. All rights reserved

The Qb-SNARE Memb11 interacts specifically with Arf1 in the Golgi apparatus of Arabidopsis thaliana

The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are critical for the function of the secretory pathway. The SNARE Memb11 is involved in membrane trafficking at the ER-Golgi interface. The aim of the work was to decipher molecular mechanisms acting in Memb11-mediated ER-Golgi traffic. In mammalian cells, the orthologue of Memb11 (membrin) is potentially involved in the recruitment of the GTPase Arf1 at the Golgi membrane. However molecular mechanisms associated to Memb11 remain unknown in plants. Memb11 was detected mainly at the cis-Golgi and co-immunoprecipitated with Arf1, suggesting that Arf1 may interact with Memb11. This interaction of Memb11 with Arf1 at the Golgi was confirmed by in vivo BiFC (Bimolecular Fluorescence Complementation) experiments. This interaction was found to be specific to Memb11 as compared to either Memb12 or Sec22. Using a structural bioinformatic approach, several sequences in the N-ter part of Memb11 were hypothesized to be critical for this interaction and were tested by BiFC on corresponding mutants. Finally, by using both in vitro and in vivo approaches, we determined that only the GDP-bound form of Arf1 interacts with Memb11. Together, our results indicate that Memb11 interacts with the GDP-bound form of Arf1 in the Golgi apparatus. © 2015 The Author

TEG4-2c scFv production process. A: Fed batch fermentation history plot.

<p>Stirring, pO₂ and OD₆₀₀ values are plotted versus time during the cultivation of <i>P</i>. <i>pastoris</i> in BMGY medium. Cultures were induced with methanol at t = 0 (24 h after starting the batch phase) during the fed batch phase the methanol was added every 12 h or 6h (black arrows) to a final concentration of 0.6%. The average values are shown with error bars representing the standard deviation (n = 5). 1 OD₆₀₀ unit was equivalent to 0.29 mg/mL dry weight. <b>B: Dot-blot analysis of supernatants from recombinant <i>P</i>. <i>pastoris</i> culture</b>. Fifty microliters samples from non-induced culture (NI) and from day 1 to day 5 induced cultures (I1d to I5d) were undiluted (a) or diluted (b = 1:10; c = 1:50) and blotted on a nitrocellulose membrane. The recombinant TEG4-2c scFv were detected with the Anti-6His antibody and revealed by colorimetric analysis.</p

IMAC Purification of scFv TEG4-2c produced and secreted by <i>P</i>. <i>pastoris</i>.

<p>Data are standardized for 1 L culture media; the values are the mean of 7 independent experiments ± SD values.</p