Technical refolding of proteins: Do we have freedom to operate?

Expression as inclusion bodies in Escherichia coli is a widely used method for the large-scale production of therapeutic proteins that do not require post-translational modifications. High expression yields and simple recovery steps of inclusion bodies from the host cells are attractive features industrially. However, the value of an inclusion body-based process is dominated by the solubilization and refolding technologies. Scale-invariant technologies that are economical and applicable for a wide range of proteins are requested by industry. The main challenge is to convert the denatured protein into its native conformation at high yields. Refolding competes with misfolding and aggregation. Thus, the yield of native monomer depends strongly on the initial protein concentrations in the refolding solution. Reasonable yields are attained at low concentrations (≤0.1 mg/mL). However, large buffer tanks and time-consuming concentration steps are required. We attempt to answer the question of the extent to which refolding of proteins is protected by patents. Low-molecular mass additives have been developed to improve refolding yields through the stabilization of the protein in solution and shielding hydrophobic patches. Progress has been made in the field of high-pressure renaturation and on-column refolding. Mixing times of the denatured protein in the refolding buffer have been reduced using newly developed devices and the introduction of specific mixers. Concepts of continuous refolding have been introduced to reduce tank sizes and increase yields. Some of the patents covering refolding of proteins will soon expire or have already expired. This gives more freedom to operate.     

A study of the occurrence of regulator secondary structures in globular proteins

The distribution of regular secondary structures, viz. α-helices and β-strands, along the length of over 70 properties whose secondary structural details have been reported, has been analysed. The occurrence of these regular structures tends to be a maximum at the N- and C-termini. Our analysis suggests that both these free ends could possibly serve as nucleating centers for secondary structures and could play an important role in the folding of proteins.     

Tissue-Characteristic Expression of Mouse Proteome

The mouse is a valuable model organism for biomedical research. Here, we established a comprehensive spectral library and the data-independent acquisition–based quantitative proteome maps for 41 mouse organs, including some rarely reported organs such as the cornea, retina, and nine paired organs. The mouse spectral library contained 178,304 peptides from 12,320 proteins, including 1678 proteins not reported in previous mouse spectral libraries. Our data suggested that organs from the nervous system and immune system expressed the most distinct proteome compared with other organs. We also found characteristic protein expression of immune-privileged organs, which may help understanding possible immune rejection after organ transplantation. Each tissue type expressed characteristic high-abundance proteins related to its physiological functions. We also uncovered some tissue-specific proteins which have not been reported previously. The testis expressed highest number of tissue-specific proteins. By comparison of nine paired organs including kidneys, testes, and adrenal glands, we found left organs exhibited higher levels of antioxidant enzymes. We also observed expression asymmetry for proteins related to the apoptotic process, tumor suppression, and organ functions between the left and right sides. This study provides a comprehensive spectral library and a quantitative proteome resource for mouse studies.     

Activation of caspase-9 and its influencing factors in beef during conditioning

To study the activation of caspase-9 and its potential influence in conditioning, longissimus thoracis (LT), semitendinosus (STN) and psoas minor (PMi) muscles were used to analyze the ratio of pro-apoptotic bax to anti-apoptotic bcl-2 in fresh tissues and observe the changes in ATP, cytosolic cytochrome c and caspase-9 activity levels during storage at 4°C. Caspase-9 activity at 5 h is higher than the activity at 0 and 24 h in the muscles (P<0.001). The ATP content decreased between 0 and 3 h, between 8 and 14 h in the PMi and LT muscles (P<0.0001), whereas between 0 and 5 h, between 8 and 14 h in the STN muscle (P<0.0001). There is 60.2%, 55.3% and 43.1% available ATP in the STN, LT and PMi muscles at 5 h, respectively. The cytosolic cytochrome c level increased during 5 and 24 h storage in the LT and PMi muscles (P<0.0001), during 5 and 96 h in the STN muscle (P<0.0001). The cytosolic cytochrome c at 24 h (P<0.001) and ratio of bax to bcl-2 (P<0.05) was higher in the PMi than in other muscles. We concluded that the increase in cytosolic cytochrome c and available intracellular ATP should be responsible for the increase in caspase-9 activity; the activation of caspase-9 could be limited by the subsequent depletion of ATP; the postmortem release level of cytochrome c could be determined by the ratio of bax to bcl-2 in fresh tissues.     

Comparative Study of Seed Protein Profiles in the Genus Pisum

Seed protein profiles of 24 wild and cultivated taxa of Pisum have been compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis. No consistent differences were detected either among wild taxa or between wild and cultivated taxa. This shows that Pisum forms a single-species complex on the basis of seed protein profiles.     

Structures and target RNA preferences of the RNA-binding protein family of IGF2BPs: An overview

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Phosphoproteome Profiling of the Receptor Tyrosine Kinase MuSK Identifies Tyrosine Phosphorylation of Rab GTPases

Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed 2 decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, agonist antibodies function independently of its coreceptor low-density lipoprotein receptor–related protein 4 to delay the onset of muscle denervation in mouse models of ALS. Here, we performed dose–response and time-course experiments on myotubes to systematically compare site-specific phosphorylation downstream of each agonist. Remarkably, both agonists elicited similar intracellular responses at known and newly identified MuSK signaling components. Among these was inducible tyrosine phosphorylation of multiple Rab GTPases that was blocked by MuSK inhibition. Importantly, mutation of this site in Rab10 disrupts association with its effector proteins, molecule interacting with CasL 1/3. Together, these data provide in-depth characterization of MuSK signaling, describe two novel MuSK inhibitors, and expose phosphorylation of Rab GTPases downstream of receptor tyrosine kinase activation in myotubes.     

A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer–Associated Stellate Cell Small Extracellular Vesicles

Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor-derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography–tandem mass spectrometry. Most of the 1481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1–like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy.