Structural and Functional Characterization of Recombinant Human Serum Transferrin Secreted from<i>Pichia pastoris</i>

Mizutani, Kimihiko; Hashimoto, Kazuhiko; Takahashi, Nobuyuki; Hirose, Masaaki; Aibara, Shigeo; Mikami, Bunzo

doi:10.1271/bbb.90635

Cited by 15 publications

(5 citation statements)

References 32 publications

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“…Another commonly used expression system for recombinant proteins is the eukaryote yeast, Pichia pastoris . While offering several advantages (high yields and low cost), for unknown reasons very little full-length hTF was successfully produced in this system until quite recently [17]. In contrast, the use of common baker’s yeast, Saccharomyces cerevisiae , has produced homogenous non-glycosylated hTF in higher yields (~1.5 g/L) than previously reported in any yeast system [18].…”

Section: Introductionmentioning

confidence: 99%

Biochemical and structural characterization of recombinant human serum transferrin from rice (Oryza sativa L.)

Steere

Bobst²,

Zhang

et al. 2012

Journal of Inorganic Biochemistry

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The Fe3+ binding protein human serum transferrin (hTF) is well known for its role in cellular iron delivery via the transferrin receptor (TFR). A new application is the use of hTF as a therapy and targeted drug delivery system for a number of diseases. Recently, production of hTF in plants has been reported; such systems provide a relatively inexpensive, animal-free (eliminating potential contamination by animal pathogens) method to produce large amounts of recombinant proteins for such biopharmaceutical applications. Specifically, the production of Optiferrin™ (hTF produced in rice, Oryza sativa, from InVitria) has been shown to yield large amounts of functional protein for use in culture medium for cellular iron delivery to promote growth. In the present work we describe further purification (by gel filtration) and characterization of hTF produced in rice (purified Optiferrin™) to determine its suitability in biopharmaceutical applications. The spectral, mass spectrometric, urea gel and kinetic analysis shows that purified Optiferrin™ is similar to recombinant nonglycosylated N-His tagged hTF expressed by baby hamster kidney cells and/or serum derived glycosylated hTF. Additionally, in a competitive immunoassay, iron-loaded Optiferrin™ is equivalent to iron-loaded N-His hTF in its ability to bind to the soluble portion of the TFR immobilized in an assay plate. As an essential requirement for any functional hTF, both lobes of purified Optiferrin™ bind Fe3+ tightly yet reversibly. Although previously shown to be capable of delivering Fe3+ to cells, the kinetics of iron release from iron-loaded Optiferrin™/sTFR and iron-loaded N-His hTF/sTFR complexes differ somewhat. We conclude that the purified Optiferrin™ might be suitable for consideration in biopharmaceutical applications.

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Section: Introductionmentioning

confidence: 99%

Biochemical and structural characterization of recombinant human serum transferrin from rice (Oryza sativa L.)

Steere

Bobst²,

Zhang

et al. 2012

Journal of Inorganic Biochemistry

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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%

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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“…Although no effect on function was observed, analysis of the purified protein by electrospray mass spectrometry identified an O-linked glycosylation on residue Ser32, not present in the naturally derived protein (Mason et al, 1996). While the P. pastoris expression system offers several advantages (high yields and low cost), until recently no significant amount of full-length hTF has been produced in this system (Mizutani et al, 2010). Recent efforts using common baker’s yeast, Saccharomyces cerevisiae , have produced homogenous non-glycosylated hTF at significantly higher yields (~1.5 g/l) than previously observed in any yeast system (Finnis et al, 2010).…”

Section: Recombinant Expressionmentioning

confidence: 99%

Transferrin-Mediated Cellular Iron Delivery

Luck

Mason

2012

Current Topics in Membranes

130

116

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Essential to iron homeostasis is the transport of iron by the bilobal protein human serum transferrin (hTF). Each lobe (N- and C-lobe) of hTF forms a deep cleft which binds a single Fe3+. Iron-bearing hTF in the blood binds tightly to the specific transferrin receptor (TFR), a homodimeric transmembrane protein. After undergoing endocytosis, acidification of the endosome initiates the release of Fe3+ from hTF in a TFR-mediated process. Iron-free hTF remains tightly bound to the TFR at acidic pH; following recycling back to the cell surface, it is released to sequester more iron. Efficient delivery of iron is critically dependent on hTF/TFR interactions. Therefore, identification of the pH-specific contacts between hTF and the TFR is crucial. Recombinant protein production has enabled deconvolution of this complex system. The studies reviewed herein support a model in which pH-induced interrelated events control receptor-stimulated iron release from each lobe of hTF.

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Structural and Functional Characterization of Recombinant Human Serum Transferrin Secreted fromPichia pastoris

Cited by 15 publications

References 32 publications

Biochemical and structural characterization of recombinant human serum transferrin from rice (Oryza sativa L.)

Biochemical and structural characterization of recombinant human serum transferrin from rice (Oryza sativa L.)

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

Transferrin-Mediated Cellular Iron Delivery

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