Laboratory Evolution of Bacillus circulans Xylanase Inserted into Pyrococcus furiosus Maltodextrin-Binding Protein for Increased Xylanase Activity and Thermal Stability Toward Alkaline pH

Shah, Vandan; Charlton, Timothy S.; Kim, Jin Ryoun

doi:10.1007/s12010-017-2619-9

Cited by 14 publications

(11 citation statements)

References 58 publications

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“…To overcome such energetic penalty, Kim and coworkers used a thermophilic maltodextrin-binding protein from Pyrococcus furiosus as a host protein, into which various guest enzyme domains, such as exoinulinase, TEM-1 β-lactamase and xylanase, were successfully inserted. The insertional fusions led to the creation of chimeric protein complexes, where thermostability of guest enzyme domains was improved by various mechanisms [38,39,40,41,42]. In contrast, similar insertional fusion into a mesophilic maltodextrin-binding protein from E. coli lowered thermostability and expression levels of a guest enzyme domain [39].…”

Section: Thermophilic Proteins As a Scaffold For Functional Evolutionmentioning

confidence: 99%

Thermophilic Proteins as Versatile Scaffolds for Protein Engineering

Finch

Kim

2018

Microorganisms

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Literature from the past two decades has outlined the existence of a trade-off between protein stability and function. This trade-off creates a unique challenge for protein engineers who seek to introduce new functionality to proteins. These engineers must carefully balance the mutation-mediated creation and/or optimization of function with the destabilizing effect of those mutations. Subsequent research has shown that protein stability is positively correlated with “evolvability” or the ability to support mutations which bestow new functionality on the protein. Since the ultimate goal of protein engineering is to create and/or optimize a protein’s function, highly stable proteins are preferred as potential scaffolds for protein engineering. This review focuses on the application potential for thermophilic proteins as scaffolds for protein engineering. The relatively high inherent thermostability of these proteins grants them a great deal of mutational robustness, making them promising scaffolds for various protein engineering applications. Comparative studies on the evolvability of thermophilic and mesophilic proteins have strongly supported the argument that thermophilic proteins are more evolvable than mesophilic proteins. These findings indicate that thermophilic proteins may represent the scaffold of choice for protein engineering in the future.

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Section: Thermophilic Proteins As a Scaffold For Functional Evolutionmentioning

confidence: 99%

Thermophilic Proteins as Versatile Scaffolds for Protein Engineering

Finch

Kim

2018

Microorganisms

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“…Kinetic stability is related to the timescale over which an enzyme remains active . Wild‐type BCX was found to lose xylanase activity during incubation (Figure A) . Less than 5 % of the original xylanase activity of wild‐type BCX was detected after 14 days′ incubation.…”

Section: Resultsmentioning

confidence: 99%

“…The DNA coding for the resulting BCX variant was referred to as bcx‐klvfwak . For the PCR, a plasmid pET23b‐BCX, in which DNA encoding BCX was cloned, was used as a template. The PCR was carried out with 5′‐TATCT TCATA TGCAC CACCA CCACC ACCAC GCCTC CACAG ACTAC TGGCA AAA‐3′ and 5′‐AATGT ATAAC TAGTT TATCA TTTCG CCCAA AACAC CAGTT TGCCA CCGCC CCACA CTGTA ACGTT GGAAG AACCA G‐3′ as forward and reverse primers, respectively; the NdeI restriction enzyme site at the 5′‐end and the SpeI restriction enzyme site at the 3′‐end of bcx‐klvfwak are underlined.…”

Section: Methodsmentioning

confidence: 99%

“…Enzymatic hydrolysis of ONPX2 was measured under constant stirring at 40 °C in PBSA, as described previously . The ratio of k cat / K m was determined from the initial rates, as described previously . For the enzyme activity assay, the concentrations of proteins and ONPX2 were 1 and 148 μ m , respectively, unless otherwise mentioned.…”

Section: Methodsmentioning

confidence: 99%

“…Reversible thermal unfolding was monitored at 222 nm while the temperature was increased from 20 to 75 °C. To prevent irreversible aggregation during the thermal unfolding while allowing a clear CD transition, the ellipticity of protein samples (with and without 4 μ m peptides) was measured at a protein concentration of 2 μ m in the presence of urea (2.5 m ), as described previously . For CD temperature scans, a scanning rate of 0.2 °C min −1 and a response time of 16 s were chosen to minimize the scan‐rate dependencies for thermal unfolding of BCX and its variant .…”

Section: Methodsmentioning

confidence: 99%

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Amyloid Aggregation of Bacillus circulans Xylanase under Native Conditions and its Modulation by β‐Amyloid‐Derived Peptide Fragments

et al. 2018

Self Cite

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The aggregation of intrinsically disordered proteins into fibrils is implicated in many neurodegenerative diseases. Amyloid aggregation is a generic property of proteins as evidenced by globular proteins that often form amyloid aggregates under partially denaturing conditions. Recently, multiple lines of evidence have suggested that the amyloid aggregation of globular proteins can also occur under native conditions. Unfortunately, amyloid aggregation under native conditions has been demonstrated in only a handful of cases. Engineering a globular protein's amyloid aggregation might benefit from its fusion to an amyloid‐derived fragment with reduced aggregation propensity. Unfortunately, the impacts of such fragments on the amyloid aggregation under native conditions have yet to be examined. In this study, we show that a globular protein, Bacillus circulans xylanase (BCX), can aggregate to form amyloid fibrils under native conditions. When BCX was mixed with or fused to the non‐self‐aggregating fragments, KLVFWAK and ELVFWAE—which were derived from β‐amyloid (Aβ)—they modulated the BCX amyloid aggregation to differing extents. This study also provides insight into a correlation between the kinetic stability and amyloid aggregation of BCX, and supports a view that Aβ‐derived fragments can be useful for the modulating amyloid aggregation of some, though not all, proteins.

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Fusion proteins development strategies and their role as cancer therapeutic agents

Aslam,

Zulfiqar,

Hameed

et al. 2023

Biotech and App Biochem

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Cancer continues to be leading cause of morbidity and mortality despite decades of research and advancement in chemotherapy. Most tumors can be reduced via standard oncology treatments, such as chemotherapy, radiotherapy, and surgical resection, and they frequently recur. Significant progress has been made since targeted cancer therapy inception in creation of medications that exhibit improved tumor‐selective action. Particularly in preclinical and clinical investigations, fusion proteins have shown strong activity and improved treatment outcomes for a number of human cancers. Synergistically combining many proteins into one complex allows the creation of synthetic fusion proteins with enhanced characteristics or new capabilities. Signal transduction pathways are important for onset, development, and spread of cancer. As result, signaling molecules are desirable targets for cancer therapies, and significant effort has been made into developing fusion proteins that would act as inhibitors of these pathways. A wide range of biotechnological and medicinal applications are made possible by fusion of protein domains that improves bioactivities or creates new functional combinations. Such proteins may function as immune effectors cell recruiters to tumors or as decoy receptors for various ligands. In this review article, we have outlined the standard methods for creating fusion proteins and covered the applications of fusion proteins in treatment of cancer. This article also highlights the role of fusion proteins in targeting the signaling pathways involved in cancer for effective treatment.

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Laboratory Evolution of Bacillus circulans Xylanase Inserted into Pyrococcus furiosus Maltodextrin-Binding Protein for Increased Xylanase Activity and Thermal Stability Toward Alkaline pH

Cited by 14 publications

References 58 publications

Thermophilic Proteins as Versatile Scaffolds for Protein Engineering

Thermophilic Proteins as Versatile Scaffolds for Protein Engineering

Amyloid Aggregation of Bacillus circulans Xylanase under Native Conditions and its Modulation by β‐Amyloid‐Derived Peptide Fragments

Fusion proteins development strategies and their role as cancer therapeutic agents

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