Refolding and Activation from Bacterial Inclusion Bodies of Trypsin I from Sardine (Sardinops sagax caerulea)

2019 ◽  
Vol 26 (3) ◽  
pp. 170-175
Author(s):  
Manuel I. Carretas-Valdez ◽  
Francisco J. Cinco-Moroyoqui ◽  
Marina J. Ezquerra-Brauer ◽  
Enrique Marquez-Rios ◽  
Idania E. Quintero-Reyes ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Inclusion Bodies ◽  
Recombinant Expression ◽  
Luria Broth ◽  
Biotechnological Applications ◽  
Trypsin Activity ◽  
Biophysical Characterization ◽  
Cold Adapted ◽  
Sardinops Sagax

Background: Trypsin from fish species is considered as a cold-adapted enzyme that may find potential biotechnological applications. In this work, the recombinant expression, refolding and activation of Trypsin I (TryI) from Monterey sardine (Sardinops sagax caerulea) are reported. Methods: TryI was overexpressed in Escherichia coli BL21 as a fusion protein of trypsinogen with thioredoxin. Refolding of trypsinogen I was achieved by dialysis of bacterial inclusion bodies with a recovery of 16.32 mg per liter of Luria broth medium. Results: Before activation, the trypsinogen fusion protein did not show trypsin activity. Trypsinogen I was activated by adding 0.002 U of native TryI purified from the sardine pyloric caeca (nonrecombinant). The activated recombinant trypsin showed three times more activity than the nonrecombinant trypsin alone. Conclusion: The described protocol allowed obtaining sufficient amounts of recombinant TryI from Monterey sardine fish for further biochemical and biophysical characterization of its coldadaptation parameters.

scholarly journals Biophysical characterization of the SARS-CoV-2 E protein

2021 ◽  
Author(s):  
Aujan Mehregan ◽  
Sergio Perez-Conesa ◽  
Yuxuan Zhuang ◽  
Ahmad Elbahnsi ◽  
Diletta Pasini ◽  
...  
Keyword(s):  
Recombinant Expression ◽  
Structural Data ◽  
Md Simulations ◽  
Full Length ◽  
Coarse Grained ◽  
Biophysical Characterization ◽  
Viral Budding ◽  
Nmr Structures ◽  
E Protein

SARS-CoV-2 is the virus responsible for the COVID-19 pandemic which continues to wreak havoc across the world, over a year and a half after its effects were first reported in the general media. Current fundamental research efforts largely focus on the SARS-CoV-2 Spike protein. Since successful antiviral therapies are likely to target multiple viral components, there is considerable interest in understanding the biophysical role of its other proteins, in particular structural membrane proteins. Here, we have focused our efforts on the characterization of the full-length E protein from SARS-CoV-2, combining experimental and computational approaches. Recombinant expression of the full-length E protein from SARS-CoV-2 reveals that this membrane protein is capable of independent multimerization, possibly as a tetrameric or smaller species. Fluorescence microscopy shows that the protein localizes intracellularly, and coarse-grained MD simulations indicate it causes bending of the surrounding lipid bilayer, corroborating a potential role for the E protein in viral budding. Although we did not find robust electrophysiological evidence of ion-channel activity, cells transfected with the E protein exhibited reduced intracellular Ca2+, which may further promote viral replication. However, our atomistic MD simulations revealed that previous NMR structures are relatively unstable, and result in models incapable of ion conduction. Our study highlights the importance of using high-resolution structural data obtained from a full-length protein to gain detailed molecular insights, and eventually permitting virtual drug screening.

scholarly journals Recombinant expression, purification, and biophysical characterization of the transmembrane and membrane proximal domains of HIV-1 gp41

2014 ◽  
Vol 23 (11) ◽  
pp. 1607-1618 ◽  
Author(s):  
Zhen Gong ◽  
Sarah A. Kessans ◽  
Lusheng Song ◽  
Katerina Dörner ◽  
Ho-Hsien Lee ◽  
...  
Keyword(s):  
Recombinant Expression ◽  
Biophysical Characterization ◽  
Hiv 1

Recombinant expression and characterization of a novel cold-adapted type I pullulanase for efficient amylopectin hydrolysis

2020 ◽  
Vol 313 ◽  
pp. 39-47 ◽  
Author(s):  
Shi-Yu Zhang ◽  
Ze-Wang Guo ◽  
Xiao-Ling Wu ◽  
Xiao-Yang Ou ◽  
Min-Hua Zong ◽  
...  
Keyword(s):  
Recombinant Expression ◽  
Type I ◽  
Cold Adapted

scholarly journals Recombinant expression and characterization of Solanum tuberosum aspartic protease

SURG Journal ◽  
2009 ◽  
Vol 3 (1) ◽  
pp. 19-25
Author(s):  
Lauren Agro ◽  
Brian Bryska ◽  
Rickey Yada
Keyword(s):  
Solanum Tuberosum ◽  
Disulfide Bonds ◽  
Inclusion Bodies ◽  
Signal Sequence ◽  
Recombinant Expression ◽  
Aspartic Proteinase ◽  
Wild Type ◽  
Sds Page ◽  
Hemoglobin Degradation

Unique to aspartic proteinases from plants are the presence of approximately 100 amino acid regions, which are usually excised during activation of the zymogen. These sequences are termed ‘Plant-Specific Inserts’ and are implicated in membrane interactions of their parent enzymes, including vacuolar targeting and host defense. The need to further characterize the structure-function rol (s) of plant-specific inserts stimulated the current study of the characterization of Solanum tuberosum AP (StAP). Recombinant expression of wild-type StAP resulted in the 54 kDa protein being visualized by SDS-PAGE analysis with a protein yield of 0.03%. A protein purification factor could not be established since activation of the protein at pH 2.2, 3.0, 3.7 and 5.5 was not achieved as evidenced by a lack of change in band patterns on SDS-PAGE as well as acidification and hemoglobin degradation assays. To potentially improve enzyme folding and activation ability, two mutants, (1) lacking the pre-signal sequence and (2) lacking both the signal sequence and the prosegment, were designed, sub-cloned, and expressed. Both products proved to be insoluble and inactive. New constructs were designed for the expression of StAP inclusion bodies for insoluble expression and subsequent re-folding of the protein. Additionally, CysAla mutations for each PSI Cys residue were made to investigate the rol (s) of plant-specific insert disulfide bonds in plant aspartic proteinase enzyme folding and structure. All PSI cysteine mutations (eight mutants) were successfully created using QuickChange MutagenesisTM

scholarly journals Biochemical and biophysical characterization of four EphB kinase domains reveals contrasting thermodynamic, kinetic and inhibition profiles

2013 ◽  
Vol 33 (3) ◽  
Author(s):  
Ross C. Overman ◽  
Judit E. Debreczeni ◽  
Caroline M. Truman ◽  
Mark S. McAlister ◽  
Teresa K. Attwood
Keyword(s):  
Tyrosine Kinases ◽  
Recombinant Expression ◽  
Biological Significance ◽  
Kinase Domain ◽  
Cell Contact ◽  
Inhibitor Binding ◽  
Binding Properties ◽  
Biophysical Characterization ◽  
Cellular Processes

The Eph (erythropoietin-producing hepatocellular carcinoma) B receptors are important in a variety of cellular processes through their roles in cell-to-cell contact and signalling; their up-regulation and down-regulation has been shown to have implications in a variety of cancers. A greater understanding of the similarities and differences within this small, highly conserved family of tyrosine kinases will be essential to the identification of effective therapeutic opportunities for disease intervention. In this study, we have developed a route to production of multi-milligram quantities of highly purified, homogeneous, recombinant protein for the kinase domain of these human receptors in Escherichia coli. Analyses of these isolated catalytic fragments have revealed stark contrasts in their amenability to recombinant expression and their physical properties: e.g., a >16°C variance in thermal stability, a 3-fold difference in catalytic activity and disparities in their inhibitor binding profiles. We find EphB3 to be an outlier in terms of both its intrinsic stability, and more importantly its ligand-binding properties. Our findings have led us to speculate about both their biological significance and potential routes for generating EphB isozyme-selective small-molecule inhibitors. Our comprehensive methodologies provide a template for similar in-depth studies of other kinase superfamily members.

scholarly journals Controlled Formation of Catalytic and Emissive Gold Nanoclusters with a Synthetic Self-Assembled Protein Toroid

2020 ◽  
Author(s):  
David Bouzada ◽  
Ester Polo ◽  
Elena López ◽  
Yolanda Pérez ◽  
Cristian Vidal ◽  
...  
Keyword(s):  
Chemical Synthesis ◽  
Self Assembly ◽  
Solid Phase ◽  
Recombinant Expression ◽  
Solid Phase Peptide Synthesis ◽  
Gold Nanoclusters ◽  
Building Blocks ◽  
Biophysical Characterization ◽  
Self Assembled

<div>Self-assembled proteins are privileged building blocks for the bottom-up organization of matter at the nanoscale. However, since most proteins are very large, they have to be produced by recombinant expression, which is less versatile and flexible than chemical synthesis. Here, we show that we can bridge the potential of proteins for nanofabrication with the simplicity and versatility of solid-phase peptide synthesis by relying on the self-assembly of the viral protein gp23.1, a small 50-residue protein that oligomerizes in solution to form a stable toroidal hexamer. We report the chemical synthesis and basic biophysical characterization of a gp23.1 mutant and show that its self-assembled hexamer templates the formation of highly monodisperse gold nanoclusters of about 1.3 nm inside its central cavity. The resulting nanoclusters show catalytic activity in cycloisomerization reactions as well as luminescence emission. This work demonstrates the versatility of this small self- assembled ring protein for a variety of nanotechnological applications.</div>

scholarly journals Controlled Formation of Catalytic and Emissive Gold Nanoclusters with a Synthetic Self-Assembled Protein Toroid

2020 ◽  
Author(s):  
David Bouzada ◽  
Ester Polo ◽  
Elena López ◽  
Yolanda Pérez ◽  
Cristian Vidal ◽  
...  
Keyword(s):  
Chemical Synthesis ◽  
Self Assembly ◽  
Solid Phase ◽  
Recombinant Expression ◽  
Solid Phase Peptide Synthesis ◽  
Gold Nanoclusters ◽  
Building Blocks ◽  
Biophysical Characterization ◽  
Self Assembled

<div>Self-assembled proteins are privileged building blocks for the bottom-up organization of matter at the nanoscale. However, since most proteins are very large, they have to be produced by recombinant expression, which is less versatile and flexible than chemical synthesis. Here, we show that we can bridge the potential of proteins for nanofabrication with the simplicity and versatility of solid-phase peptide synthesis by relying on the self-assembly of the viral protein gp23.1, a small 50-residue protein that oligomerizes in solution to form a stable toroidal hexamer. We report the chemical synthesis and basic biophysical characterization of a gp23.1 mutant and show that its self-assembled hexamer templates the formation of highly monodisperse gold nanoclusters of about 1.3 nm inside its central cavity. The resulting nanoclusters show catalytic activity in cycloisomerization reactions as well as luminescence emission. This work demonstrates the versatility of this small self- assembled ring protein for a variety of nanotechnological applications.</div>

Recombinant Expression, in Vitro Refolding, and Biophysical Characterization of the Human Glucagon-like Peptide-1 Receptor

Biochemistry ◽  
2010 ◽  
Vol 49 (36) ◽  
pp. 7956-7965 ◽  
Author(s):  
Kathrin Schröder-Tittmann ◽  
Eva Bosse-Doenecke ◽  
Steffen Reedtz-Runge ◽  
Christian Ihling ◽  
Andrea Sinz ◽  
...  
Keyword(s):  
Recombinant Expression ◽  
Biophysical Characterization ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
In Vitro Refolding
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