Protein Characterization of the Aqueous Soluble Phase of Acidified and Autolyzed Bolti Fish (Tilapia nilotica) Viscera

ABSTRACT In yeast, enzymes with β-glucanase activity are thought to be necessary in morphogenetic events that require controlled hydrolysis of the cell wall. Comparison of the sequence of the Saccharomyces cerevisiae exo-β(1,3)-glucanase Exg1 with the Schizosaccharomyces pombe genome allowed the identification of three genes that were named exg1 + (locus SPBC1105.05), exg2 + (SPAC12B10.11), and exg3 + (SPBC2D10.05). The three proteins have different localizations: Exg1 is secreted to the periplasmic space, Exg2 is a membrane protein, and Exg3 is a cytoplasmic protein. Characterization of the biochemical activity of the proteins indicated that Exg1 and Exg3 are active only against β(1,6)-glucans while no activity was detected for Exg2. Interestingly, Exg1 cleaves the glucans with an endohydrolytic mode of action. exg1 + showed periodic expression during the cell cycle, with a maximum coinciding with the septation process, and its expression was dependent on the transcription factor Sep1. The Exg1 protein localizes to the septum region in a pattern that was different from that of the endo-β(1,3)-glucanase Eng1. Overexpression of Exg2 resulted in an increase in cell wall material at the poles and in the septum, but the putative catalytic activity of the protein was not required for this effect.

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Genomic, Tissue Expression, and Protein Characterization of pCLCA1, a Putative Modulator of Cystic Fibrosis in the Pig

Journal of Histochemistry & Cytochemistry ◽

10.1369/jhc.2009.954594 ◽

2009 ◽

Vol 57 (12) ◽

pp. 1169-1181 ◽

Cited By ~ 15

Author(s):

Stephanie Plog ◽

Lars Mundhenk ◽

Nikolai Klymiuk ◽

Achim D. Gruber

Keyword(s):

Cystic Fibrosis ◽

Tissue Expression ◽

Protein Characterization

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Optimization of Expression Condition, Two Dimensional And Melting Point-Based Characterization of Recombinant Human Interferon Alpha-2a Fusion and Non Fusion Forms

ANNALES BOGORIENSES ◽

10.14203/ann.bogor.2018.v22.n2.57-64 ◽

2018 ◽

Vol 22 (2) ◽

pp. 57

Author(s):

Ratih Asmana Ningrum ◽

Widdya Kusuma Wardhani ◽

Ike Wahyuni ◽

Apon Zaenal Mustopa

Keyword(s):

Interferon Alpha ◽

Protein Characterization ◽

Protein Yield ◽

Size Exclusion ◽

Human Interferon ◽

Two Dimensional ◽

Cancer Treatments ◽

Sds Page ◽

Bca Assay

Recombinant Human Interferon Alpha-2a (rhIFNα-2a) is a therapeutic protein that used in hepatitis and cancer treatments. In our previous research, we developed higher molecular weight of the protein through human serum albumin fusion. The fusion and non fusion form of rhIFNα-2a were produced in Pichia pastoriswith 86 kDa and 19 kDa in size respectively. In previous research, protein yield was not reproducible due to unoptimized expression conditions. This reseach was aimed to optimize expression condition process and to characterize the fusion and non fusion forms of rhIFNα-2a. The parameters to observe in overproduction include nutrient (media and methanol concentration) and non nutrient (temperature andincubation period). Affinity and size exclusion cromatographicwere compared in protein purification. BCA assay was used to determine quantity of protein. Protein characterization was conducted using two-dimensional SDS PAGE and denaturation analyses. The optimal condition of expression was achieved using complex media with 1% of methanol for 3 day incubation period at 25°C. The protein yield was reproducible and higher comparing to previous research. Affinity chromatography resulted in higher purity of the proteins comparing to size exclusions. Characterization using two dimensional gel analysis revealed that isoelectric point of rhIFNα-2a is 6.5 for fusion form and 6.0 for non fusion form. The melting points of fusion protein were 56°C and 62°C whilst that of non fusion was 56°C.

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A Novel Karyoskeletal Protein: Characterization of Protein NO145, the Major Component of Nucleolar Cortical Skeleton inXenopus Oocytes

Molecular Biology of the Cell ◽

10.1091/mbc.12.12.3904 ◽

2001 ◽

Vol 12 (12) ◽

pp. 3904-3918 ◽

Cited By ~ 8

Author(s):

Sandra Kneissel ◽

Werner W. Franke ◽

Joseph G. Gall ◽

Hans Heid ◽

Sonja Reidenbach ◽

...

Keyword(s):

Divalent Cations ◽

Protein Characterization ◽

Electron Microscopic Level ◽

Expression Library ◽

Electron Microscopic ◽

Egg Formation ◽

Complex Protein ◽

Calculated Mass ◽

Synaptonemal Complex Protein

The nucleolus is a ubiquitous, mostly spheroidal nuclear structure of all protein-synthesizing cells, with a well-defined functional compartmentalization. Although a number of nonribosomal proteins involved in ribosome formation have been identified, the elements responsible for the shape and internal architecture of nucleoli are still largely unknown. Here, we report the molecular characterization of a novel protein, NO145, which is a major and specific component of a nucleolar cortical skeleton resistant to high salt buffers. The amino acid sequence of this polypeptide with a SDS-PAGE mobility corresponding to M r 145,000 has been deduced from a cDNA clone isolated from a Xenopus laevis ovary expression library and defines a polypeptide of 977 amino acids with a calculated mass of 111 kDa, with partial sequence homology to a synaptonemal complex protein, SCP2. Antibodies specific for this protein have allowed its recognition in immunoblots of karyoskeleton-containing fractions of oocytes from differentXenopus species and have revealed its presence in all stages of oogenesis, followed by a specific and rapid degradation during egg formation. Immunolocalization studies at the light and electron microscopic level have shown that protein NO145 is exclusively located in a cage-like cortical structure around the entire nucleolus, consisting of a meshwork of patches and filaments that dissociates upon reduction of divalent cations. We propose that protein NO145 contributes to the assembly of a karyoskeletal structure specific for the nucleolar cortex of the extrachromosomal nucleoli ofXenopus oocytes, and we discuss the possibility that a similar structure is present in other cells and species.

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Molecular cloning and protein characterization of swine 4-1BB

Veterinary Immunology and Immunopathology ◽

10.1016/j.vetimm.2013.01.016 ◽

2013 ◽

Vol 153 (1-2) ◽

pp. 35-44 ◽

Cited By ~ 1

Author(s):

Xinxin Zhao ◽

Huali Su ◽

Xiaoxi Huang ◽

Lili Xie ◽

Zhengzhu Liu ◽

...

Keyword(s):

Molecular Cloning ◽

Protein Characterization

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Arabidopsis thaliana β1,2-xylosyltransferase: an unusual glycosyltransferase with the potential to act at multiple stages of the plant N-glycosylation pathway

Biochemical Journal ◽

10.1042/bj20042091 ◽

2005 ◽

Vol 388 (2) ◽

pp. 515-525 ◽

Cited By ~ 42

Author(s):

Peter BENCÚR ◽

Herta STEINKELLNER ◽

Barbara SVOBODA ◽

Jan MUCHA ◽

Richard STRASSER ◽

...

Keyword(s):

Catalytic Activity ◽

Substrate Specificity ◽

Metal Ion ◽

Catalytic Domain ◽

Protein Characterization ◽

Reaction Products ◽

Glycosylation Sites ◽

Glycosylation Pathway ◽

Multiple Stages

XylT (β1,2-xylosyltransferase) is a unique Golgi-bound glycosyltransferase that is involved in the biosynthesis of glycoprotein-bound N-glycans in plants. To delineate the catalytic domain of XylT, a series of N-terminal deletion mutants was heterologously expressed in insect cells. Whereas the first 54 residues could be deleted without affecting the catalytic activity of the enzyme, removal of an additional five amino acids led to the formation of an inactive protein. Characterization of the N-glycosylation status of recombinant XylT revealed that all three potential N-glycosylation sites of the protein are occupied by N-linked oligosaccharides. However, an unglycosylated version of the enzyme displayed substantial catalytic activity, demonstrating that N-glycosylation is not essential for proper folding of XylT. In contrast with most other glycosyltransferases, XylT is enzymatically active in the absence of added metal ions. This feature is not due to any metal ion directly associated with the enzyme. The precise acceptor substrate specificity of XylT was assessed with several physiologically relevant compounds and the xylosylated reaction products were subsequently tested as substrates of other Golgi-resident glycosyltransferases. These experiments revealed that the substrate specificity of XylT permits the enzyme to act at multiple stages of the plant N-glycosylation pathway.

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