scholarly journals A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hitomi Nakamura ◽  
Moeka Yoshikawa ◽  
Naoko Oda-Ueda ◽  
Tadashi Ueda ◽  
Takatoshi Ohkuri
Keyword(s):  
Thermal Stability ◽  
Pichia Pastoris ◽  
Protein Stability ◽  
Disulfide Bond ◽  
Binding Activity ◽  
Comprehensive Analysis ◽  
The Other ◽  
Constant Domain ◽  
Intermolecular Disulfide Bond ◽  
Sds Page

AbstractGenerally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bond can be introduced into the Fab’s constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the F130C(H):Q124C(L), F174C(H):S176C(L), V177C(H):Q160C(L), F174C(H):S162C(L), F130C(H):S121C(L), and A145C(H):F116C(L) mutants mostly formed intermolecular disulfide bond. All these mutants showed increased thermal stability compared to that of Fab without intermolecular disulfide bond. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C(H):F118C(L) mutant showed only a slight decrease in binding activity and β-helix content, owing to the exertion of adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals that the introduction of intermolecular disulfide bond in the Fab’s constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.

scholarly journals A Comprehensive Analysis of Novel Disulfide Bond Introduction Site into the Constant Domain of Human Fab

2021 ◽  
Author(s):  
Hitomi Nakamura ◽  
Moeka Yoshikawa ◽  
Naoko Oda-Ueda ◽  
Tadashi Ueda ◽  
Takatoshi Ohkuri
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Binding Activity ◽  
Comprehensive Analysis ◽  
The Other ◽  
Thermal Stabilities ◽  
Constant Domain ◽  
Intermolecular Disulfide Bond ◽  
Sds Page ◽  
Intermolecular Disulfide Bonds

Abstract Generally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bonds can be introduced into the Fab’s constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the H: F130C-L: Q124C, H: F174C-L: S176C, H: V177C-L: Q160C, H: F174C-L: S162C, H: F130C-L: S121C, and H: A145C-L: F116C mutants mostly formed intermolecular disulfide bonds. All these mutants showed increased thermal stabilities compared to those without intermolecular disulfide bonds. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C-F118C mutant showed only a slight decrease in binding activity and β-helix content, owing to adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals the introduction of intermolecular disulfide bonds in the Fab’s constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.

scholarly journals Enhancement of protein stability by an additional disulfide bond designed in human neuroglobin

RSC Advances ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 4172-4179 ◽  
Author(s):  
Hai-Xiao Liu ◽  
Lianzhi Li ◽  
Xin-Zhi Yang ◽  
Chuan-Wan Wei ◽  
Hui-Min Cheng ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Protein Stability ◽  
Disulfide Bond ◽  
Ph Stability ◽  
Structural Alterations

A disulfide bond of Cys120 and Cys15 was rationally designed in human neuroglobin (Ngb) by A15C mutation, which caused minimal structural alterations, whereas enhanced both chemical and pH stability, with a thermal stability higher than 100 °C.

XYLANASE, AN ENVIRONMENTALLY FRIENDLY BLEACHING AGENT FOR PULP AND PAPER: Characterization And Stabilization Study Of Bacillus licheniformis I-5 CRUDE Xylanase

2018 ◽  
Vol 7 (3) ◽  
Author(s):  
Budiasih Wahyuntari., dkk
Keyword(s):  
Thermal Stability ◽  
Bacillus Licheniformis ◽  
Hot Spring ◽  
Sodium Dodecyl ◽  
Luria Broth ◽  
Crude Enzyme ◽  
Half Time ◽  
Triton X100 ◽  
Sds Page ◽  
Xylanolytic Enzymes

Isolate I-5 was isolated from Ciseeng hot spring, West Java and was identified as Bacillus licheniformis I-5. The isolate produces extracellular xylanolytic enzymes on Oatspelt containing Luria broth agar medium. Optimal activity of the crude enzyme was  observed at 50ºC and pH 7. The effect of sodium dodecyl sulphate, b-mercaptoethanol and Triton-X100 were observed. Incubating the crude enzyme in 1.5% SDS and 1.5% b-mercaptoethanol at 50oC for 90 minutes then adding Triton-X100 at final concentration of 3.5% for 45 minutes only reduced 5.75% of the initial enzyme activity. SDS/PAGE and zymogram analysis showed that at least two xylanolytic enzymes presence in the crude enzyme. The molecular weight of the enzyme was estimated about 127 and 20kD. The enzyme hydrolysed xylan into xylobiose, xylotriose and other longer xylooligosaccharides. Thermal stability of the crude enzyme was observed at 50, 60, and 70oC and pH 7 and 8. The results showed that the half time of the crude enzyme incubated at 50, 60, and 70oC pH 7 was 2 hours 55 minutes; 2 hours 33 minutes and 1 hour 15 minutes respectively. The half time at 50, 60 and 70oC, pH 8 was 2 hours 48 minutes; 1 hour 22 minutes and 1 hour 9 minutes respectively.keywords: Xilanase, Bacillus licheniformis I-5, thermal stability

scholarly journals Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Laura Navone ◽  
Thomas Vogl ◽  
Pawarisa Luangthongkam ◽  
Jo-Anne Blinco ◽  
Carlos H. Luna-Flores ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Phytic Acid ◽  
Disulfide Bond ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
E Coli ◽  
Biochemical Characterisation ◽  
Disulfide Bond Isomerase ◽  
Microbial Systems ◽  
Protein Disulfide

Abstract Background Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market. Results In this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in kcat and KM for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme. Conclusions Overall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes.

scholarly journals Effect of Pre-Fermentative Maceration and Fining Agents on Protein Stability, Macromolecular, and Phenolic Composition of Albariño White Wines: Comparative Efficiency of Chitosan, k-Carrageenan and Bentonite as Heat Stabilisers

Foods ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 608
Author(s):  
Inma Arenas ◽  
Miguel Ribeiro ◽  
Luís Filipe-Ribeiro ◽  
Rafael Vilamarim ◽  
Elisa Costa ◽  
...  
Keyword(s):  
Phenolic Compounds ◽  
Protein Stability ◽  
White Wine ◽  
The Other ◽  
White Wines ◽  
Comparative Efficiency ◽  
Fining Agents ◽  
Protein Instability ◽  
Related Proteins ◽  
Protein Stabilisation

In this work, the effect of pre-fermentative skin maceration (PFSM) on the chemical composition of the macromolecular fraction, polysaccharides and proteins, phenolic compounds, chromatic characteristics, and protein stability of Albariño monovarietal white wines was studied. PFSM increased the extraction of phenolic compounds and polysaccharides and reduced the extraction of pathogenesis-related proteins (PRPs). PFSM wine showed significantly higher protein instability. Sodium and calcium bentonites were used for protein stabilisation of wines obtained with PFSM (+PFSM) and without PFSM (−PFSM), and their efficiencies compared to fungal chitosan (FCH) and k-carrageenan. k-Carrageenan reduced the content of PRPs and the protein instability in both wines, and it was more efficient than sodium and calcium bentonites. FCH was unable to heat stabilise both wines, and PRPs levels remained unaltered. On the other hand, FCH decreased the levels of wine polysaccharides by 60%. Sodium and calcium bentonite also decreased the levels of wine polysaccharides although to a lower extent (16% to 59%). k-Carrageenan did not affect the wine polysaccharide levels. Overall, k-carrageenan is suitable for white wine protein stabilisation, having a more desirable impact on the wine macromolecular fraction than the other fining agents, reducing the levels of the wine PRPs without impacting polysaccharide composition.

Special Items: A Descriptive Analysis

2011 ◽  
Vol 25 (3) ◽  
pp. 511-536 ◽  
Author(s):  
Peter M. Johnson ◽  
Thomas J. Lopez ◽  
Juan Manuel Sanchez
Keyword(s):  
Firm Value ◽  
Temporal Frequency ◽  
Descriptive Analysis ◽  
Financial Statements ◽  
Comprehensive Analysis ◽  
The Other ◽  
Future Earnings ◽  
Special Items ◽  
Increasing Function ◽  
Frequency Magnitude

SYNOPSIS We provide a comprehensive analysis of special items and the characteristics of the firms that recognize them. Our analysis reveals that the temporal frequency, magnitude, and persistence of special items has increased significantly in the last 30 years, and that such increases are primarily driven by negative special items. More recently, however, our evidence is consistent with both a decline in frequency and magnitude of negative special items. On the other hand, we find that the frequency of reporting of positive special items, which remained relatively constant through 2002, has increased in more recent years. We also find strong evidence that subsequent special item reporting is an increasing function of the frequency of “prior” special item reporting. Using a random subsample of firms reporting special items, we document that 22 percent of the amounts reported in Compustat do not reconcile with the amounts reported on the firms' actual financial statements. Our comprehensive analysis should be of interest to regulators, academics, and managers interested in the implications of special items on firm-related consequences such as future earnings and firm value. Our examination can also serve as a catalyst for researchers interested in extending this important area of inquiry.

Compact Hardware Implementations of MISTY1 Block Cipher

2017 ◽  
Vol 27 (03) ◽  
pp. 1850037 ◽  
Author(s):  
Yasir ◽  
Ning Wu ◽  
Xiaoqiang Zhang
Keyword(s):  
Low Power ◽  
Block Cipher ◽  
Comprehensive Analysis ◽  
The Other ◽  
Key Generation ◽  
Round Function ◽  
Other Hand ◽  
Hardware Implementations ◽  
Covered Area

This paper proposes compact hardware implementations of 64-bit NESSIE proposed MISTY1 block cipher for area constrained and low power ASIC applications. The architectures comprise only one round MISTY1 block cipher algorithm having optimized FO/FI function by re-utilizing S9/S7 substitution functions. A focus is also made on efficient logic implementations of S9 and S7 substitution functions using common sub-expression elimination (CSE) and parallel AND/XOR gates hierarchy. The proposed architecture 1 generates extended key with independent FI function and is suitable for MISTY1 8-rounds implementation. On the other hand, the proposed architecture 2 uses a single FO/FI function for both MISTY1 round function as well as extended key generation and can be employed for MISTY1 [Formula: see text] rounds. To analyze the performance and covered area for ASICs, Synopsys Design Complier, SMIC 0.18[Formula: see text][Formula: see text]m @ 1.8[Formula: see text]V is used. The hardware constituted 3041 and 2331 NAND gates achieving throughput of 171 and 166 Mbps for 8 rounds implementation of architectures 1 and 2, respectively. Comprehensive analysis of proposed designs is covered in this paper.

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