Construction, Investigation and Application of TEV Protease Variants with Improved Oxidative Stability

Bayar, Enkhtuya; Ren, Yuanyuan; Chen, Yinghua; Hu, Yafang; Zhang, Shuncheng; Yu, Xuelian; Fan, Jun

doi:10.4014/jmb.2106.06075

Cited by 6 publications

(8 citation statements)

References 37 publications

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“…The reason was attributed to that the cysteine residues in one TEVp variant molecule were linked to another molecule via the mis-matched disul de bonds, and C151 was responsible for catalysis. Mutation of the cysteine residues increases the TEVp oxidative stability [30]. In contrast, three TEVp variants with C110 and/or C130S mutations exhibited the relatively high cleavage activity (Fig.…”

Section: Extracellular Production and Puri Cation Of The Hana1 Tagged...mentioning

confidence: 93%

“…In each fusion protein, the TEVp recognition sequence (ENLYFQG↓S, tevS) was incorporated. The plasmids for expressing the mutated TEVp including TEVp1 (S219V), TEVp2 (L56V/S135G/S219V), TEVp3 (T17S/N68D/I77V/S219V), TEVp4 (T17S/L56V/N68D/I77V/S135G/ S219V), and TEVp5 (T17S/L56V/N68D/E106G/I77V/S135G/ S219V), TEVp6 (C151A in TEVp4), TEVp7 (C110S in TEVp4) TEVp8 (C130S in TEVp4), TEVp9 (C110S and C130S in TEVp4), the GST-EmGFP as a TEVp substrate were constructed in our laboratory [29,30]. The bacteriophage ΦX174 gene E encoding the lytic protein (LyE) was inserted into the pBAD33.1 plasmid to yield pBAD-LyE for controlling the gene expression under the control of the P BAD promoter [31].…”

Section: Methodsmentioning

confidence: 99%

“…The TEVp cleavage activity was analyzed. The GST-EmGFP in crude extract, and the puri ed TEVp variant fusion was mixed with mass ratio of 10: 1 to react at 30°C for 1 h. The mixture was incubated with SDS-PAGE loading buffer at 40°C for 30 min, and subjected to SDS-PAGE analysis for in-gel uorescence display [30]. The EmGFP with the N-terminal cellulose-binding module (CBM) tag was immobilized on the regenerated amorphous cellulose with buffer A or washed with buffer A containing 5 mM dithiothreitol (DTT).…”

Section: Rapid Separation and Cleavage Activity Assay Of The Tevp Var...mentioning

confidence: 99%

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Extracellular production, purification and tag-removal of the N-terminally annexin- and ELP-tagged fluorescent proteins

Chen,

Li,

et al. 2024

Preprint

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Background Rapid and cost-effective purification of the target protein for extracellular production in Escherichia coli is still challenge. Previously, we identified that human annexin A1 as a N-terminal fusion tag for Ca2+-dependent phase transition to simply, rapidly and cost-effectively purify three fluorescent proteins including emerald green fluorescent protein (EmGFP), red fluorescent protein mCherry, and flavin-binding cyan-green fluorescent protein.Results When the phage lytic protein was induced later, the annexin A1 tagged EmGFP was leaked into the culture, but purification efficiency was relatively low. Pre-overexpression of Bacillus cereus phospholipase C facilitated intracellular production of the fusion protein, and purified fusion protein showed the purity higher than other two fluorescent protein fusions. Using the co-expression system, the elastin-like polypeptide (ELP) tagged three fluorescent proteins via extracellular production were also purified in revisable protein precipitation. The yield of the purified annexin A1 tagged protein was comparable to that of the purified ELP tagged one. The silica-binding peptide tagged annexin A1-EmGFP bound to silica particles, and the ELP tagged mCherry strongly bound to phenyl sepharose was efficient for column-dependent purification. The extracellular nine tobacco etch virus protease variants with the annexin A1 tag were purified and the cleavage activity was assayed. Using the purified protease variant with the highest activity, the purification tag was removed in solution, or by on-resin cleavage of the immobilized annexin A1 or ELP tagged EmGFP. The soluble annexin A1-EmGFP with the Bacillus amyloliquefaciens alpha-amylase signal peptide was poorly produced in Bacillus subtilis, and the fusion protein with the α-factor signal peptide was located in intracellular Pichia pastoris.Conclusions The annexin A1 or ELP fusions in the culture were purified by revise transition cycles. On-resin cleavage facilitated removal of the reagents for protein purification, and fusion tag. However, the annexin A1-EmGFP fused the correspondent signal peptides displayed poor secretion efficiency in B. subtilis and P. pastoris. The platform will be used for simply and cost-effectively purifying the target proteins with industrial and clinical values without cell disruption process, and rapidly testifying the activity of the engineered enzyme variants.

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Section: Extracellular Production and Puri Cation Of The Hana1 Tagged...mentioning

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Section: Rapid Separation and Cleavage Activity Assay Of The Tevp Var...mentioning

confidence: 99%

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Extracellular production, purification and tag-removal of the N-terminally annexin- and ELP-tagged fluorescent proteins

Chen,

Li,

et al. 2024

Preprint

Self Cite

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“…Furthermore, not all disulfide bridges of the BbPETase CD formed upon expression, which could be attributed to the E. coli Rosetta gami-B (DE3) strain. Expression of proteins with disulfide bridges by the E. coli Rosetta gami-B (DE3) strain may be not very effective [25]. Therefore, it may adversely affect the correct formation of disulfide bridges in BbPETase CD and its variants.…”

Section: Rational Design Of Disulfide Bridges and Protein Expressionmentioning

confidence: 99%

Rational Design of Disulfide Bridges in BbPETaseCD for Enhancing the Enzymatic Performance in PET Degradation

2023

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Polyethylene terephthalate (PET) is one of the most prevalent transparent thermoplastics. It is commonly utilized due to its low cost and high durability. With the massive accumulation of waste PET, however, serious environmental pollution has become a global problem. Compared to traditional chemical degradation, biodegradation of PET catalyzed by PET hydrolase (PETase) is more environmentally friendly and energy-efficient. BbPETaseCD from the Burkholderiales bacterium is a PETase that shows favorable properties for application in the biodegradation of PET. To enhance the enzymatic performance of this enzyme, this work focuses on the rational design of disulfide bridges in BbPETaseCD. We utilized two computational algorithms to predict the probable disulfide-bridge mutations in BbPETaseCD, and five variants were acquired from the computations. Among these, the N364C/D418C variant with one additional disulfide bond showed higher expression than the wild-type enzyme (WT) and the best enzymatic performance. The melting temperature (Tm) of the N364C/D418C variant presented an increase of 14.8 °C over that of WT (56.5 °C), indicating that the additional disulfide bond significantly raised the thermodynamic stability of the enzyme. Kinetic experiments at different temperatures also demonstrated the thermal stability increase of the variant. The variant also showed significantly increased activity over WT when using bis(hydroxyethyl) terephthalate (BHET) as the substrate. More remarkably, the N364C/D418C variant exhibited approximately an 11-fold increase over the WT enzyme in the long-term (14 days) degradation of PET films. The results prove that the rationally designed disulfide bond significantly improved the enzymatic performance of the enzyme for PET degradation.

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“…With the biotechnological use of an enzyme comes a need to improve or adjust its properties. Variants with improved solubility and yield, , reduced self-cleavage, and improved oxidative stability as well as immobilization strategies were reported . However, the biggest interest lies in the improvement of TEVp catalytic activity, since the wild type is slow compared to other widely used proteases that commonly implement serine as a nucleophile.…”

Section: Introductionmentioning

confidence: 99%

Between Protein Fold and Nucleophile Identity: Multiscale Modeling of the TEV Protease Enzyme–Substrate Complex

Zlobin

Golovin

2022

ACS Omega

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The cysteine protease from the tobacco etch virus (TEVp) is a well-known and widely utilized enzyme. TEVp's chymotrypsin-like fold is generally associated with serine catalytic triads that differ in terms of a reaction mechanism from the most well-studied papain-like cysteine proteases. The question of what dominates the TEVp mechanism, nucleophile identity, or structural composition has never been previously addressed. Here, we use enhanced sampling multiscale modeling to uncover that TEVp combines the features of two worlds in such a way that potentially hampers its activity. We show that TEVp cysteine is strictly in the anionic form in a free enzyme similar to papain. Peptide binding shifts the equilibrium toward the nucleophile′s protonated form, characteristic of chymotrypsin-like proteases, although the cysteinyl anion form is still present and interconversion is rapid. This way cysteine protonation generates enzyme states that are a diversion from the most effective course of action, with only 13.2% of Michaelis complex sub-states able to initiate the reaction. As a result, we propose an updated view on the reaction mechanism catalyzed by TEVp. We also demonstrate that AlphaFold is able to construct protease−substrate complexes with high accuracy. We propose that our findings open a way for its industrious use in enzymological tasks. Unique features of TEVp discovered in this work open a discussion on the evolutionary history and trade-offs of optimizing serine triad-associated folds to cysteine as a nucleophile.

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Construction, Investigation and Application of TEV Protease Variants with Improved Oxidative Stability

Cited by 6 publications

References 37 publications

Extracellular production, purification and tag-removal of the N-terminally annexin- and ELP-tagged fluorescent proteins

Extracellular production, purification and tag-removal of the N-terminally annexin- and ELP-tagged fluorescent proteins

Rational Design of Disulfide Bridges in BbPETaseCD for Enhancing the Enzymatic Performance in PET Degradation

Between Protein Fold and Nucleophile Identity: Multiscale Modeling of the TEV Protease Enzyme–Substrate Complex

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