Structural and Functional Characterization of Ovotransferrin Produced by<i>Pichia pastoris</i>

Mizutani, Kimihiko; Okamoto, Ikuko; Fujita, Kiyotaka; Yamamoto, Kenji; Hirose, Masaaki

doi:10.1271/bbb.68.376

Cited by 13 publications

(17 citation statements)

References 34 publications

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“…NicaTf was expressed in P. pastoris cells. This system has been used to express other transferrins (29)(30)(31). The purification is illustrated in SI Fig.…”

Section: Resultsmentioning

confidence: 99%

On the evolutionary significance and metal-binding characteristics of a monolobal transferrin fromCiona intestinalis

Tinoco

Peterson

Lucchese

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…NicaTf was expressed in P. pastoris cells. This system has been used to express other transferrins (29)(30)(31). The purification is illustrated in SI Fig.…”

Section: Resultsmentioning

confidence: 99%

On the evolutionary significance and metal-binding characteristics of a monolobal transferrin fromCiona intestinalis

Tinoco

Peterson

Lucchese

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…26,27,49,50,54,55) In general, the construction of an expression system using P. pastoris is time consuming; the expression plasmid is usually constructed using the traditional method (manipulation using enzymes and amplification in E. coli), and then the constructed plasmid is inserted into the genome of P. pastoris. Cregg et al developed the first P. pastoris expression system using autonomous plasmids with PARS1 or PARS2 (replication sequence derived from the genome of P. pastoris).…”

Section: (B)mentioning

confidence: 99%

“…26,54,55) Recently, I and my collaborators introduced the PARS1 sequence into the Life Technologies plasmids and developed new autonomous plasmids (such as p9KPrAHS) compatible with the Life Technologies plasmids (Fig. 4(A)).…”

Section: (B)mentioning

confidence: 99%

“…I am currently trying to develop a high-throughput method for construction of cell surface protein production system of P. pastoris, which will be applicable for biotechnological applications, such as collection of metals by recombinant transferrins, 13,25,26) using a plasmid with the gene for cell wall protein PIR3.…”

Section: (B)mentioning

confidence: 99%

“…20,25) I and my collaborators constructed a P. pastoris expression system of human serum transferrin and chicken transferrin by a traditional method. 13,26,27) Recombinant proteins were secreted into media and had N-linked high-mannose carbohydrate chains. Both proteins had tertiary structures similar to native proteins, and bound two irons as native proteins.…”

mentioning

confidence: 99%

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High-throughput plasmid construction using homologous recombination in yeast: its mechanisms and application to protein production for X-ray crystallography

Mizutani

2015

Bioscience, Biotechnology, and Biochemistry

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To cite this article: Kimihiko Mizutani (2015) High-throughput plasmid construction using homologous recombination in yeast: its mechanisms and application to protein production for X-ray crystallography, Bioscience, Biotechnology, and Biochemistry Award ReviewHigh-throughput plasmid construction using homologous recombination in yeast: its mechanisms and application to protein production for X-ray crystallography Homologous recombination is a system for repairing the broken genomes of living organisms by connecting two DNA strands at their homologous sequences. Today, homologous recombination in yeast is used for plasmid construction as a substitute for traditional methods using restriction enzymes and ligases. This method has various advantages over the traditional method, including flexibility in the position of DNA insertion and ease of manipulation. Recently, the author of this review reported the construction of plasmids by homologous recombination in the methanol-utilizing yeast Pichia pastoris, which is known to be an excellent expression host for secretory proteins and membrane proteins. The method enabled high-throughput construction of expression systems of proteins using P. pastoris; the constructed expression systems were used to investigate the expression conditions of membrane proteins and to perform X-ray crystallography of secretory proteins. This review discusses the mechanisms and applications of homologous recombination, including the production of proteins for X-ray crystallography.Key words: homologous recombination; plasmid construction; Pichia pastoris; expression system; X-ray crystallographyTo repair their broken genomes, living organisms have a system connecting two DNA strands at their similar (homologous) sequences.1-3) This system is called homologous recombination, and it is employed for genetic recombination during meiosis. In recent years, homologous recombination in yeast has been used for plasmid construction (such as insertion of a target gene into a plasmid) and gene isolation (such as transformation-associated recombination cloning to directly capture genomic loci from Saccharomyces cerevisiae).4,5) Unlike with traditional methods using restriction enzymes and ligases, the method for plasmid construction using homologous recombination can insert a DNA fragment into an appropriate and favorable position of a plasmid. The regions of homology used for recombination need not be at the ends of the linearized plasmid.4) For example, using a plasmid with a green fluorescence protein (GFP) in the region downstream of the promoter, homologous recombination has been applied to high-throughput construction of a yeast S. cerevisiae expression system of GFP membrane protein fusions.6,7) This system has been efficiently used to investigate the expression, purification, and crystallization conditions of membrane proteins. As another example, a method was developed for converting a common Escherichia coli vector to an E. coli yeast shuttle vector to enable the construction of plasmid by ho...

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Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2003–2004

Harvey

2008

Mass Spectrometry Reviews

100

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This review is the third update of the original review, published in 1999, on the application of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings the topic to the end of 2004. Both fundamental studies and applications are covered. The main topics include methodological developments, matrices, fragmentation of carbohydrates and applications to large polymeric carbohydrates from plants, glycans from glycoproteins and those from various glycolipids. Other topics include the use of MALDI MS to study enzymes related to carbohydrate biosynthesis and degradation, its use in industrial processes, particularly biopharmaceuticals and its use to monitor products of chemical synthesis where glycodendrimers and carbohydrate-protein complexes are highlighted.

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Structural and Functional Characterization of Ovotransferrin Produced byPichia pastoris

Cited by 13 publications

References 34 publications

On the evolutionary significance and metal-binding characteristics of a monolobal transferrin fromCiona intestinalis

On the evolutionary significance and metal-binding characteristics of a monolobal transferrin fromCiona intestinalis

High-throughput plasmid construction using homologous recombination in yeast: its mechanisms and application to protein production for X-ray crystallography

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2003–2004

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