scholarly journals Separation of native and truncated forms of poliovirus protease 3C produced in Escherichia coli

1993 ◽  
Vol 290 (3) ◽  
pp. 797-800 ◽  
Author(s):  
L Polgár ◽  
F Erdélyi ◽  
E Hajnal ◽  
M Löw ◽  
L Gráf ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Initiation Site ◽  
Full Length ◽  
Size Exclusion ◽  
Truncated Form ◽  
Buffer Solution ◽  
Diluted Solution ◽  
Exclusion Chromatography ◽  
Internal Initiation

Poliovirus protease 3C is a cysteine enzyme that is essential for the processing of the viral precursor polyprotein containing structural proteins and enzymes, including the protease itself. We have constructed the plasmid pSD/PV3C which produced protease 3C as inclusion bodies when expressed in Escherichia coli. In addition to the full-length protease, a truncated form was also generated, starting from an internal initiation site (Met-27). The enzyme was renatured by dilution of a 6 M guanidinium chloride solution of the inclusion bodies, and the proteins were precipitated from the diluted solution with ammonium sulphate. By extracting the precipitate with a buffer solution, the full-length enzyme could be completely separated from its N-terminally truncated form. Size-exclusion chromatography of the extracted protease 3C resulted in an active enzyme which appeared homogeneous by SDS/PAGE. For measuring the activity of the protease, a spectrofluorimetric method was devised to monitor the hydrolysis continuously, which is simpler and more precise than the h.p.l.c. technique used previously.

scholarly journals Enhancement of the Structural Stability of Full-Length Clostridial Collagenase by Calcium Ions

2012 ◽  
Vol 78 (16) ◽  
pp. 5839-5844 ◽  
Author(s):  
Naomi Ohbayashi ◽  
Noriko Yamagata ◽  
Masafumi Goto ◽  
Kimiko Watanabe ◽  
Youhei Yamagata ◽  
...  
Keyword(s):  
Melting Point ◽  
Structural Stability ◽  
Conformational Changes ◽  
Full Length ◽  
Size Exclusion ◽  
Content Type ◽  
Protein Size ◽  
Exclusion Chromatography ◽  
Domain Arrangement ◽  
Thermal Melting

ABSTRACTThe clostridial collagenases G and H are multidomain proteins. For collagen digestion, the domain arrangement is likely to play an important role in collagen binding and hydrolysis. In this study, the full-length collagenase H protein fromClostridium histolyticumwas expressed inEscherichia coliand purified. The N-terminal amino acid of the purified protein was Ala31. The expressed protein showed enzymatic activity against azocoll as a substrate. To investigate the role of Ca2+in providing structural stability to the full-length collagenase H, biophysical measurements were conducted using the recombinant protein. Size exclusion chromatography revealed that the Ca2+chelation by EGTA induced interdomain conformational changes. Dynamic light scattering measurements showed an increase in the percent polydispersity as the Ca2+was chelated, suggesting an increase in protein flexibility. In addition to these conformational changes, differential scanning fluorimetry measurements revealed that the thermostability was decreased by Ca2+chelation, in comparison with the thermal melting point (Tm). The melting point changed from 54 to 49°C by the Ca2+chelation, and it was restored to 54°C by the addition of excess Ca2+. These results indicated that the interdomain flexibility and the domain arrangement of full-length collagenase H are reversibly regulated by Ca2+.

scholarly journals VirB8 homolog TraE from plasmid pKM101 forms a hexameric ring structure and interacts with the VirB6 homolog TraD

2018 ◽  
Vol 115 (23) ◽  
pp. 5950-5955 ◽  
Author(s):  
Bastien Casu ◽  
Charline Mary ◽  
Aleksandr Sverzhinsky ◽  
Aurélien Fouillen ◽  
Antonio Nanci ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Secretion System ◽  
High Molecular Mass ◽  
Full Length ◽  
Size Exclusion ◽  
Cross Linking ◽  
X Ray ◽  
Exclusion Chromatography ◽  
Plasmid Pkm101 ◽  
Membrane Bound

Type IV secretion systems (T4SSs) are multiprotein assemblies that translocate macromolecules across the cell envelope of bacteria. X-ray crystallographic and electron microscopy (EM) analyses have increasingly provided structural information on individual T4SS components and on the entire complex. As of now, relatively little information has been available on the exact localization of the inner membrane-bound T4SS components, notably the mostly periplasmic VirB8 protein and the very hydrophobic VirB6 protein. We show here that the membrane-bound, full-length version of the VirB8 homolog TraE from the plasmid pKM101 secretion system forms a high-molecular-mass complex that is distinct from the previously characterized periplasmic portion of the protein that forms dimers. Full-length TraE was extracted from the membranes with detergents, and analysis by size-exclusion chromatography, cross-linking, and size exclusion chromatography (SEC) multiangle light scattering (MALS) shows that it forms a high-molecular-mass complex. EM and small-angle X-ray scattering (SAXS) analysis demonstrate that full-length TraE forms a hexameric complex with a central pore. We also overproduced and purified the VirB6 homolog TraD and show by cross-linking, SEC, and EM that it binds to TraE. Our results suggest that TraE and TraD interact at the substrate translocation pore of the secretion system.

scholarly journals Efficient independent activity of a monomeric, monofunctional dehydroquinate synthase derived from the N-terminus of the pentafunctional AROM protein of Aspergillus nidulans

1994 ◽  
Vol 301 (1) ◽  
pp. 297-304 ◽  
Author(s):  
J D Moore ◽  
J R Coggins ◽  
R Virden ◽  
A R Hawkins
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Aspergillus Nidulans ◽  
Chelating Agents ◽  
Size Exclusion ◽  
Functional Domain ◽  
N Terminus ◽  
Exclusion Chromatography ◽  
Km Value ◽  
Dehydroquinate Synthase

The dehydroquinate synthase (DHQ synthase) functional domain from the pentafunctional AROM protein of Aspergillus nidulans has previously been overproduced in Escherichia coli [van den Hombergh, Moore, Charles and Hawkins (1992) Biochem J. 284, 861-867]. We now report the purification of this domain to homogeneity and subsequent characterization. The monofunctional DHQ synthase was found to retain efficient catalytic activity when compared with the intact pentafunctional AROM protein of Neurospora crassa [Lambert, Boocock and Coggins (1985) Biochem J. 226, 817-829]. The apparent kcat. was estimated to be 8 s-1, and the apparent Km values for NAD+ and 3-deoxy-D-arabino-heptulosonate phosphate (DAHP) were 3 microM and 2.2 microM respectively. These values are similar to those reported for the intact N. crassa enzyme, except that the apparent Km for NAD+ reported here is 15-fold higher. The monofunctional DHQ synthase domain is inactivated by treatment with chelating agents in the absence of substrates and is re-activated by the addition of metal ions; among those tested, Zn2+ gave the highest kcat./Km value. The enzyme is inactivated by diethyl pyrocarbonate; both the substrate, DAHP, and the product phosphate protected against inactivation. Size-exclusion chromatography suggested an M(r) of 43,000 for the monofunctional domain, indicating that it is monomeric and compactly folded. The c.d. spectrum confirmed that the domain has a compact globular conformation; the near-u.v. c.d. of zinc- and cobalt-reactivated domains were superimposable.

scholarly journals Enhanced oligomerization of full-length RAGE by synergy of the interaction of its domains

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Alexander Moysa ◽  
Dietmar Hammerschmid ◽  
Roman H. Szczepanowski ◽  
Frank Sobott ◽  
Michal Dadlez
Keyword(s):  
Analytical Ultracentrifugation ◽  
Vascular Diseases ◽  
Native Mass Spectrometry ◽  
Full Length ◽  
Size Exclusion ◽  
Glycation End Products ◽  
Exclusion Chromatography ◽  
Hydrogen Deuterium

AbstractThe pattern recognition receptor RAGE (receptor for advanced glycation end-products) transmits proinflammatory signals in several inflammation-related pathological states, including vascular diseases, cancer, neurodegeneration and diabetes. Its oligomerization is believed to be important in signal transduction, but RAGE oligomeric structures and stoichiometries remain unclear. Different oligomerization modes have been proposed in studies involving different truncated versions of the extracellular parts of RAGE. Here, we provide basic characterization of the oligomerization patterns of full-length RAGE (including the transmembrane (TM) and cytosolic regions) and compare the results with oligomerization modes of its four truncated fragments. For this purpose, we used native mass spectrometry, analytical ultracentrifugation, and size-exclusion chromatography coupled with multi-angle light scattering. Our results confirm known oligomerization tendencies of separate domains and highlight the enhanced oligomerization properties of full-length RAGE. Mutational analyses within the GxxxG motif of the TM region show sensitivity of oligomeric distributions to the TM sequence. Using hydrogen–deuterium exchange, we mapped regions involved in TM-dependent RAGE oligomerization. Our data provide experimental evidence for the major role of the C2 and TM domains in oligomerization, underscoring synergy among different oligomerization contact regions along the RAGE sequence. These results also explain the variability of obtained oligomerization modes in RAGE fragments.

scholarly journals PTD-mediated delivery of α-globin chain into Κ-562 erythroleukemia cells and α-thalassemic (HBH) patients’ RBCs ex vivo in the frame of Protein Replacement Therapy

2021 ◽  
Vol 28 (1) ◽  
Author(s):  
Androulla N. Miliotou ◽  
Dionysia Papagiannopoulou ◽  
Efthymia Vlachaki ◽  
Martina Samiotaki ◽  
Dimitra Laspa ◽  
...  
Keyword(s):  
Replacement Therapy ◽  
Inclusion Bodies ◽  
Ex Vivo ◽  
Research Work ◽  
Size Exclusion ◽  
Globin Chain ◽  
Therapy Approach ◽  
Globin Chains ◽  
Exclusion Chromatography

Abstract Background α-Thalassemia, a congenital hemoglobinopathy, is characterized by deficiency and/or reduced levels of α-globin chains in serious forms of α-thalassemia (HbH disease/Hb Bart’s). This research work deals with a Protein Replacement Therapy approach in order to manage α-thalassemia manifestations, caused by the excess of β-globin chain into HbH RBCs. The main goal was to produce the recombinant human α-globin chain in fusion with TAT, a Protein Transduction Domain, to ex vivo deliver it into HbH patients RBCs, to replace the endogenous missing α-globin chain. Results Cloning of the α-globin coding sequence, fused to the nucleotide sequence of TAT peptide was conducted and the human recombinant fusion proteins, 10xHis-XaSITE-α-globin-HA and 10xHis-XaSITE-TAT-α-globin-HA were produced. The ability of human recombinant 10xHis-XaSITE-α-globin-HA to interact in vitro with the previously produced 10xHis-XaSITE-TAT-β-globin-HA and form α-/β-globin heterodimers, was assessed and confirmed by size exclusion chromatography. The recombinant 10xHis-XaSITE-TAT-α-globin-HA was successfully delivered into human proerythroid K-562 cells, during the preliminary transduction evaluation experiments. Finally, the recombinant, TAT-fused α-globin was successfully transduced into RBCs, derived from HbH patients and reduced the formation of HbH-Inclusion Bodies, known to contain harmful β4-globin chain tetramers. Conclusions Our data confirm the successful ex vivo transduction of recombinant α-globin chains in HbH RBCs to replace the missing a-globin chain and reduce the HbH-inclusion bodies, seen in α-thalassemias. These findings broaden the possibility of applying a Protein Replacement Therapy approach to module sever forms of α-thalassemia, using recombinant α-globin chains, through PTD technology.

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