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Full-length recombinant human SCF1-165 is more thermostable than the truncated SCF1-141 form.

ABSTRACT: Human stem cell factor initiates a diverse array of cellular responses, including hematopoiesis, cell proliferation, differentiation, migration and survival. To explore the relationship between its structure and function, we produced recombinant soluble human stem cell factor1-165 (wild type) and human stem cell factor1-141 (C-terminal truncated) in a yeast expression system and compared their biological activities and thermal stabilities. The biological activity of the two proteins was measured as a function of TF-1 cell viability and effects on downstream signaling targets after incubation. We found that these proteins enhanced cell viability and downstream signaling to a similar extent, in a dose-dependent manner. The biological activity of recombinant human stem cell factor1-165 was significantly greater than that of recombinant human stem cell factor1-141 after heating the proteins (100 ng/mL) at 25-110°C for 10 minutes (P<0.05 for all temperatures). In addition, circular dichroism spectral analysis indicated that ?-sheet structures were altered in recombinant human stem cell factor1-141 but not recombinant human stem cell factor1-165 after heating at 90°C for 15 or 30 min. Molecular modeling and limited proteolytic digestion were also used to compare the thermo stability between human stem cell factor1-165 and human stem cell factor1-141. Together, these data indicate that stem cell factor1-165 is more thermostable than stem cell factor1-141.

SUBMITTER: Weng YP

PROVIDER: S-EPMC4111497 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Full-length recombinant human SCF1-165 is more thermostable than the truncated SCF1-141 form.

Weng Yui-Ping YP Ku Wen-Yen WY Wu Ming-Han MH Tsai Ya-Li YL Chen Chi-Yu CY Kuo Chun-An CA Huang Lynn L H LL

PloS one 20140725 7

Human stem cell factor initiates a diverse array of cellular responses, including hematopoiesis, cell proliferation, differentiation, migration and survival. To explore the relationship between its structure and function, we produced recombinant soluble human stem cell factor1-165 (wild type) and human stem cell factor1-141 (C-terminal truncated) in a yeast expression system and compared their biological activities and thermal stabilities. The biological activity of the two proteins was measured ...[more]

PMID: 25061857

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Project description:BACKGROUND: ?-1,3-Glucanases catalyze the hydrolysis of glucan polymers containing ?-1,3-linkages. These enzymes are of great biotechnological, agricultural and industrial interest. The applications of ?-1,3-glucanases is well established in fungal disease biocontrol, yeast extract production and wine extract clarification. Thus, the identification and characterization of novel ?-1,3-glucanases with high catalytic efficiency and stability is of particular interest. RESULTS: A ?-1,3-glucanase gene designated PglA was cloned from a newly isolated strain Paenibacillus sp. S09. The gene PglA contained a 2631-bp open reading frame encoding a polypeptide of 876 amino acids which shows 76% identity with the ?-1,3-glucanase (BglH) from Bacillus circulans IAM1165. The encoded protein PglA is composed of a signal peptide, an N-terminal leader region, a glycoside hydrolase family 16 (GH16) catalytic domain and a C-terminal immunoglobulin like (Ig-like) domain. The Escherichia coli expression system of PglA and five truncated derivatives containing one or two modules was constructed to investigate the role of catalytic and non-catalytic modules. The pH for optimal activity of the enzymes was slightly affected (pH 5.5-6.5) by the presence of different modules. However, the temperature for optimal activity was strongly influenced by the C-terminal domain and ranged from 50 to 60°C. Deletion of C-terminal domain resulted in obviously enhancing enzymatic thermostability. Specific activity assay indicated that PglA specifically hydrolyzes ?-1,3-glucan. Insoluble ?-1,3-glucan binding and hydrolysis were boosted by the presence of N-and C-terminal domains. Kinetic analysis showed that the presence of N-and C-terminus enhances the substrate affinity and catalytic efficiency of the catalytic domain toward laminarin. Carbohydrate-binding assay directly confirmed the binding capabilities of the N-and C-terminal domains. CONCLUSIONS: This study provides new insight into the impacts of non-catalytic modules on enzymatic properties of ?-1,3-glucanase. Activity comparison of full-length PglA and truncated forms revealed the negative effect of C-terminal region on thermal stability of the enzyme. Both the N-and C-terminal domains exerted strong binding activity toward insoluble ?-1,3-glucan, and could be classified into CBM families.

Project description:IntroductionBacillus anthracis is the causative agent for the lethal disease anthrax, primarily affecting animals and humans in close contact with an infected host. The pathogenicity of B. anthracis is attributed to the secreted exotoxins and their outer capsule. The host cell-binding exotoxin component "protective antigen" (PA) is reported to be a potent vaccine candidate. The aim of our study is to produce several PA constructs and analyze their vaccine potential.MethodsWe have designed the various subunit, PA-based recombinant proteins, i.e., full-length Protective antigen (PA-FL), C-terminal 63 kDa fragment (PA63), Protective antigen domain 1-domain 4 chimeras (PA-D1-4) and protective antigen domain 4 (PA-D4) and analyzed their vaccine potential with different human-compatible adjuvants in the mouse model. We have optimized the process and successfully expressed our recombinant antigens as soluble proteins, except full-length PA. All the recombinant antigen formulations with three different adjuvants i.e., Addavax, Alhydrogel, and Montanide ISA 720, were immunized in different mouse groups. The vaccine efficacy of the formulations was analyzed by mouse serum antigen-specific antibody titer, toxin neutralization assay, and survival analysis of mouse groups challenged with a lethal dose of B. anthracis virulent spores.ResultsWe have demonstrated that the PA-FL addavax and PA63 addavax formulations were most effective in protecting spore-challenged mice and serum from the mice immunized with PAFL addavax, PA-FL alhydrogel, PA63 addavax, and PA63 alhydrogel formulations were equivalently efficient in neutralizing the anthrax lethal toxin. The higher levels of serum Th1, Th2, and Th17 cytokines in PA-FL addavax immunized mice correspond to the enhanced protection provided by the formulation in challenged mice.DiscussionWe have demonstrated that the PA-FL addavax and PA63 addavax formulations exhibit equivalent efficiency as vaccine formulation both in a mouse model of anthrax and mammalian cell lines. However, PA63 is a smaller antigen than PA-FL and more importantly, PA63 is expressed as a soluble protein in E. coli, which imparts a translational advantage to PA63-based formulation. Thus, the outcome of our study has significant implications for the development of protective antigen-based vaccine formulations for human use against the lethal disease anthrax.

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Dataset Information

Full-length recombinant human SCF1-165 is more thermostable than the truncated SCF1-141 form.

Publications

Full-length recombinant human SCF1-165 is more thermostable than the truncated SCF1-141 form.

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