Improved expression, purification and characterization of VPR, a cold active subtilisin-like serine proteinase and the effects of calcium on expression and stability

Óskarsson, Kristinn Ragnar; Kristjánsson, Magnús M.

doi:10.1016/j.bbapap.2018.11.010

Cited by 6 publications

(18 citation statements)

References 31 publications

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“…VPR ΔC is expressed as a preproenzyme, containing a N-terminal intramolecular chaperone that is cleaved off during maturation leaving a 28 kDa active protease with an α/β-fold 11,13,34 . This maturation leaves a kinetically stable enzyme in a process that may be similar to what has been described for α-lytic protease 11,13,25,34,35 . During this maturation the calcium binding site 3 (Ca3) (Fig.…”

Section: Discussionmentioning

confidence: 67%

“…All proline variants were successfully overexpressed in the E. coli strain Lemo21 and purified to homogeneity following the reformed production and purification protocol 25 . Thus, all single and the N3P/I5P proline variants 26 have been recharacterized with respect to activity, thermal inactivation (T 50% ) and the melting of the secondary structure (T m (CD) ).…”

Section: Resultsmentioning

confidence: 99%

“…(2020) 10:1045 | https://doi.org/10.1038/s41598-020-57873-3 www.nature.com/scientificreports www.nature.com/scientificreports/ The most important calcium binding sites for the stability are believed to be calcium binding site 1 (Ca1) (Fig. 1), mainly coordinated by the conserved Asp196 residue and Ca3 located in the N-terminal region 25,30 . The third calcium binding site is the low affinity Ca2 site thought to mainly serve as a defence against exogenous proteolysis 25 .…”

Section: Discussionmentioning

confidence: 99%

“…The N-terminus, where both these mutations are located also harbours the calcium 3 binding site, which has been suggested to be highly important for the overall stability of the enzyme (Fig. 7) 25 . Combined with the high activation energy of the second transition, this could indicate that the intermediate still retains the calcium-binding site 3.…”

Section: Stabilitymentioning

confidence: 99%

“…MD-simulation at different temperatures have suggested that the initiation point of unfolding is helix D 29 . Helix D is proximal to the Ca1 site, a site that is important for the stability of the structure 25,30,31 . Thus, by restricting movements and enforce interactions to that part of the protein might stabilize it at higher temperatures.…”

Section: Stabilitymentioning

confidence: 99%

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Thermostabilization of VPR, a kinetically stable cold adapted subtilase, via multiple proline substitutions into surface loops

Óskarsson

Sævarsson

Kristjánsson

2020

Sci Rep

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protein stability is a widely studied topic, there are still aspects however that need addressing. in this paper we examined the effects of multiple proline substitutions into loop regions of the kinetically stable proteinase K-like serine protease VpR, using the thermostable structural homologue AQUi as a template. four locations for proline substitutions were chosen to imitate the structure of AQUi. Variants were produced and characterized using differential scanning calorimetry (DSC), circular dichroism (CD), steady state fluorescence, acrylamide fluorescence quenching and thermal inactivation experiments. The final product VPR Δc _N3P/I5P/N238P/T265P was greatly stabilized which was achieved without any noticeable detrimental effects to the catalytic efficiency of the enzyme. This stabilization seems to be derived from the conformation restrictive properties of the proline residue in its ability to act as an anchor point and strengthen pre-existing interactions within the protein and allowing for these interactions to prevail when thermal energy is applied to the system. in addition, the results underline the importance of the synergy between distant local protein motions needed to result in stabilizing effects and thus giving an insight into the nature of the stability of VPR, its unfolding landscape and how proline residues can infer kinetic stability onto protein structures.

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Section: Discussionmentioning

confidence: 67%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Stabilitymentioning

confidence: 99%

Section: Stabilitymentioning

confidence: 99%

See 3 more Smart Citations

Thermostabilization of VPR, a kinetically stable cold adapted subtilase, via multiple proline substitutions into surface loops

Óskarsson

Sævarsson

Kristjánsson

2020

Sci Rep

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Versatility of subtilisin: A review on structure, characteristics, and applications

Azrin

Ali

Rahman

et al. 2022

Biotech and App Biochem

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Due to its thermostability and high pH compatibility, subtilisin is most known for its role as an additive for detergents in which it is categorized as a serine protease according to MEROPS database. Subtilisin is typically isolated from various bacterial species of the Bacillus genus such as Bacillus subtilis, B. amyloliquefaciens, B. licheniformis, and various other organisms. It is composed of 268-275 amino acid residues and is initially secreted in the precursor form, preprosubtilisin, which is composed of 29-residues signal peptide, 77-residues propeptide, and 275-residues active subtilisin. Subtilisin is known for the presence of high and low affinity calcium binding sites in its structure. Native subtilisin has general properties of thermostability, tolerance to neutral to high pH, broad specificity, and calcium-dependent stability, which contribute to the versatility of subtilisin applicability. Through protein engineering and immobilization technologies, many variants of subtilisin have been generated, which increase the applicability of subtilisin in various industries including detergent, food processing and packaging, synthesis of inhibitory peptides, therapeutic, and waste management applications.

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Gene expression and molecular characterization of recombinant subtilisin from Bacillus subtilis with antibacterial, antioxidant and anticancer properties

Shettar,

Bagewadi,

Yaraguppi

et al. 2023

International Journal of Biological Macromolecules

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Improved expression, purification and characterization of VPR, a cold active subtilisin-like serine proteinase and the effects of calcium on expression and stability

Cited by 6 publications

References 31 publications

Thermostabilization of VPR, a kinetically stable cold adapted subtilase, via multiple proline substitutions into surface loops

Thermostabilization of VPR, a kinetically stable cold adapted subtilase, via multiple proline substitutions into surface loops

Versatility of subtilisin: A review on structure, characteristics, and applications

Gene expression and molecular characterization of recombinant subtilisin from Bacillus subtilis with antibacterial, antioxidant and anticancer properties

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