Characterization of Member of DUF1888 Protein Family, Self-cleaving and Self-assembling Endopeptidase

Osipiuk, J.; Mulligan, R.; Bargassa, M.; Hamilton, John E.; Cunningham, Mark A.; Joachimiak, A.

doi:10.1074/jbc.m112.358069

Cited by 5 publications

(2 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For the cleavage we observe here, the propensity for cleavage of the Asp–Ala bond at the intein N-terminus indicates that it is catalyzed by the intein, particularly since peptide bond cleavage after Asp, in most cases, occurs at pH values below the p K a of Asp, or between particularly labile Asp–Pro bonds. ,,,− It is possible that the typical first step of splicing is promoted by shifting the equilibrium of the amide-ester rearrangement by catalytic bond strain of the amide peptide bond, by improving the electrophilicity of the carbonyl of the peptide bond by protonating the carbonyl oxygen, or by facilitating protonation of the amino leaving group either prior or concomitant to amide bond scission. Each catalytic strategy also would increase the susceptibility of the scissile N-terminal splicing junction to cleavage via an aspartic acid effect.…”

Section: Resultsmentioning

confidence: 89%

“…It is well-known that cleavage after Asp can occur at very low pH in both peptides and proteins. − ,− Given that it is apparent that our intein fusion protein can cleave in its active site downstream from a non-native Asp, we were interested in addressing two questions: Although conditions of very low pH can facilitate cleavage after Asp in peptides, is this particular cleavage dependent on the catalytic action of a folded intein? Although other inteins have shown unusual cleavage activity when flanked by Asp at their N-terminus, is the cleavage in this case clearly attributed to Asp cyclization and anhydride formation? …”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Intein-Promoted Cyclization of Aspartic Acid Flanking the Intein Leads to Atypical N-Terminal Cleavage

et al. 2017

View full text Add to dashboard Cite

Protein splicing is a post-translational reaction facilitated by an intein, or intervening protein, which involves the removal of the intein and the ligation of the flanking polypeptides, or exteins. A DNA polymerase II intein from Pyrococcus abyssi (Pab PolII intein) can promote protein splicing in vitro on incubation at high temperature. Mutation of active site residues Cys1, Gln185, and Cys+1 to Ala results in an inactive intein precursor, which cannot promote the steps of splicing, including cleavage of the peptide bond linking the N-extein and intein (N-terminal cleavage). Surprisingly, coupling the inactivating mutations to a change of the residue at the C-terminus of the N-extein (N-1 residue) from the native Asn to Asp reactivates N-terminal cleavage at pH 5. Similar "aspartic acid effects" have been observed in other proteins and peptides but usually only occur at lower pH values. In this case, however, the unusual N-terminal cleavage is abolished by mutations to catalytic active site residues and unfolding of the intein, indicating that this cleavage effect is mediated by the intein active site and the intein fold. We show via mass spectrometry that the reaction proceeds through cyclization of Asp resulting in anhydride formation coupled to peptide bond cleavage. Our results add to the richness of the understanding of the mechanism of protein splicing and provide insight into the stability of proteins at moderately low pH. The results also explain, and may help practitioners avoid, a side reaction that may complicate intein applications in biotechnology.

show abstract

Section: Resultsmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Intein-Promoted Cyclization of Aspartic Acid Flanking the Intein Leads to Atypical N-Terminal Cleavage

et al. 2017

View full text Add to dashboard Cite

show abstract

Efficient expression of nattokinase in Bacillus licheniformis: host strain construction and signal peptide optimization

Wei

Zhou

Chen

et al. 2015

Journal of Industrial Microbiology and Biotechnology

View full text Add to dashboard Cite

Nattokinase (NK) possesses the potential for prevention and treatment of thrombus-related diseases. In this study, high-level expression of nattokinase was achieved in Bacillus licheniformis WX-02 via host strain construction and signal peptides optimization. First, ten genes (mpr, vpr, aprX, epr, bpr, wprA, aprE, bprA, hag, amyl) encoding for eight extracellular proteases, a flagellin and an amylase were deleted to obtain B. licheniformis BL10, which showed no extracellular proteases activity in gelatin zymography. Second, the gene fragments of P43 promoter, Svpr, nattokinase and TamyL were combined into pHY300PLK to form the expression vector pP43SNT. In BL10 (pP43SNT), the fermentation activity and product activity per unit of biomass of nattokinase reached 14.33 FU/mL and 2,187.71 FU/g respectively, which increased by 39 and 156 % compared to WX-02 (pP43SNT). Last, Svpr was replaced with SsacC and SbprA, and the maximum fermentation activity (33.83 FU/mL) was achieved using SsacC, which was 229 % higher than that of WX-02 (pP43SNT). The maximum NK fermentation activity in this study reaches the commercial production level of solid state fermentation, and this study provides a promising engineered strain for industrial production of nattokinase, as well as a potential platform host for expression of other target proteins.

show abstract

Design and Identification of a High Efficient Formic Acid Cleavage Site For Separation of Fusion Protein

Zhang

Shi

et al. 2014

Protein J

View full text Add to dashboard Cite

The release of target protein with high efficiency and low cost from expressed fusion protein is a key requirement for commercial production of target proteins. To establish such a cleavage system, we have designed four formic acid (FA) cleavage sites C1 (DPDPDP), C2 (DPPDPP), C3 (DDDDPI) and C4 (IVDPNP), which was placed in between the E and G fusion protein. Four expression vectors were individually constructed and expressed in Escherichia coli. Purified proteins were reacted with a series of FA concentrations or under different temperatures followed by SDS-PAGE gel electrophoresis to verify the degree of cleavage efficiency. Results showed that the C2 was the most efficient site compared with the other three. After optimization of cleavage conditions for E-C2-G, the cleavage efficiently could reach as high as 87.3% within 2.5 h in 37% FA at 45 °C. Comparing with previous reports, a significant reduction (26%) of FA concentration at a lower temperature in a short duration of reaction (18 times less) was achieved. We believe the cleavage site of DPPDPP identified in this study can be used in the large-scale production of valuable fusion proteins to save the cost, time and energy.

show abstract

Characterization of Member of DUF1888 Protein Family, Self-cleaving and Self-assembling Endopeptidase

Cited by 5 publications

References 42 publications

Intein-Promoted Cyclization of Aspartic Acid Flanking the Intein Leads to Atypical N-Terminal Cleavage

Intein-Promoted Cyclization of Aspartic Acid Flanking the Intein Leads to Atypical N-Terminal Cleavage

Efficient expression of nattokinase in Bacillus licheniformis: host strain construction and signal peptide optimization

Design and Identification of a High Efficient Formic Acid Cleavage Site For Separation of Fusion Protein

Contact Info

Product

Resources

About