Molecular cloning of the chicken avidin cDNA

Gope, Mohan L.; Keinänen, R; Kristn, Paula A.; Conneely, Orla M.; Beattie, Wanda G.; Zarucki-Schulz, Tanya; O’Malley, Bert W.; Kulomaa, Markku S.

doi:10.1093/nar/15.8.3595

Cited by 73 publications

(43 citation statements)

References 48 publications

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“…The gene coding for avidin was constructed by assembling eight synthetic oligonucleotides of 89Ϫ105 bp (Primm) designed on the protein sequence reported in [2].…”

Section: Methodsmentioning

confidence: 99%

“…The interaction with biotin is non-covalent but extremely tight, the dissociation constant of about 1 fM being about 10 3 Ϫ10 6 times higher than that of a typical antigen-antibody interaction. The cDNA-derived and protein-derived sequences [2,3] and the crystallographic structures of avidin [4Ϫ6] are known. Functional avidin is a tetramer of identical subunits and each subunit is folded into an eight-stranded anti-parallel β-barrel displaying up-and-down topology.…”

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confidence: 99%

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Biochemical characterization and crystal structure of a recombinant hen avidin and its acidic mutant expressed in Escherichia coli

Nardone

Rosano

Santambrogio

et al. 1998

European Journal of Biochemistry

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The mature hen avidin encoded by a synthetic cDNA was expressed in Escherichia coli in an insoluble form. After resolubilization, renaturation and purification, a recovery of about 20 mg/l cell culture was obtained. ELISA assays indicated no apparent differences in biotin binding between the natural and recombinant avidins. In addition, an acidic avidin mutant, bearing the substitutions Lys3→Glu, Lys9→ Glu, Arg26→Asp and Arg124→Leu of four exposed basic residues, was produced. The protein, expressed and renatured as wild-type avidin, showed unaltered biotin-binding activity. The acidic pI (Ϸ5.5) and lack of aggregation of the mutant allowed easy electrophoretic analysis under non-denaturing conditions of the protein alone and of its complexes with biotin, biotinylated transferrin or peroxidase. Analysis of the sera from sensitized subjects revealed that the avidin mutant has altered antigenicity. Both recombinant avidins were crystallized and the three-dimensional structures solved by molecular replacement and refined to 0.22 nm resolution. The three-dimensional structures of the two recombinant molecules, in the absence of biotin and of glycosylation, are fully comparable with those of the natural hen avidin previously reported.Keywords : avidin; crystal structure ; mutagenesis ; recombinant protein.Avidin, a minor constituent of egg white of reptiles, amphibia and birds, is a glycosylated and positively charged protein which can bind up to four molecules of vitamin H, D-biotin [1]. The interaction with biotin is non-covalent but extremely tight, the dissociation constant of about 1 fM being about 10 3 Ϫ10 6 times higher than that of a typical antigen-antibody interaction. The cDNA-derived and protein-derived sequences [2,3] and the crystallographic structures of avidin [4Ϫ6] are known. Functional avidin is a tetramer of identical subunits and each subunit is folded into an eight-stranded anti-parallel β-barrel displaying up-and-down topology. The biotin-binding site is located in the core of the barrel, and is built by residues of the barrel itself and partly by a loop of an adjacent subunit. The interest in avidin derives mainly from several applications based on its rapid and almost irreversible binding to biotin and to practically any biotin-based molecular construct [7,8]. Avidin is used in vivo for the targeting of solid tumors [9] in applications where proteinsurface properties are determinants for proper biodistribution, serum clearance and immunological response [10]. The production of recombinant avidin and its mutated forms could increase the already remarkable versatility of this protein and may improve its properties for in vivo use.

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“…The gene coding for avidin was constructed by assembling eight synthetic oligonucleotides of 89Ϫ105 bp (Primm) designed on the protein sequence reported in [2].…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Biochemical characterization and crystal structure of a recombinant hen avidin and its acidic mutant expressed in Escherichia coli

Nardone

Rosano

Santambrogio

et al. 1998

European Journal of Biochemistry

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“…Kulomaa and co-workers [7,8] documented the cDNA of the chicken oviduct avidin gene and isolated a genomic clone. The first X-ray structure of the biotin-avidin complex reported by Bayer and co-workers [9], allowed to rationalize the strength of the biotin-avidin interaction.…”

mentioning

confidence: 99%

Expression and purification of a recombinant avidin with a lowered isoelectric point in Pichia pastoris

Zocchi

Jobé

Neuhaus

et al. 2003

Protein Expression and Purification

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A recombinant glycosylated avidin (recGAvi) with an acidic isoelectric point was expressed and secreted by the methylotrophic yeast Pichia pastoris. The coding sequence for recGAvi was de novo synthesized based on the codon usage of P. pastoris. RecGAvi is secreted at approximately 330 mg/L of culture supernatant. RecGAvi monomer displays a molecular weight of 16.5 kDa, as assessed by ESI mass spectrometry. N-terminal amino acid sequencing indicates the presence of three additional amino acids (E-A-E), which contribute to further lowering the isoelectric point to 5.4. The data presented here demonstrate that the P. pastoris system is suitable for the production of recGAvi and that the recombinant avidin displays biotin-binding properties similar to those of the hen-egg white protein.

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“…Similarly, mam-tam2A in pUC19 was digested with KpnI and XbaI and cloned into pcDNA3.1(+). The open-reading frame of the entire native avidin gene 27) was synthesized and cloned into pMA. In this case, the sequence "AAGCTTGCC" was placed just upstream of the translation initiation codon, and the sequence "GGATCC" was placed just downstream of the translation stop codon.…”

Section: Methodsmentioning

confidence: 99%

High-level expression of tamavidin 2 in human cells by codon-usage optimization

Takakura

Katayama

Nagata

2015

Bioscience, Biotechnology, and Biochemistry

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Tamavidin 2 is a fungal protein that binds to biotin with an extremely high affinity. Tamavidin 2 is superior to avidin or streptavidin in terms of its low-level non-specific binding and high-level thermal stability. However, the gene for tamavidin 2 is highly expressed in Escherichia coli but not in mammalian cells, restricting its application as an affinity tag in mammalian cells. Here, we optimized the codon usage of tamavidin 2 for human cells and found that the resultant mutant expressed tamavidin 2 at approximately 30-fold higher level compared with the native gene. The protein thus produced in human cells could be purified by iminobiotin affinity chromatography, bound tightly to biotin, and was stable at high temperature (82 °C). This powerful technology for high-level expression of tamavidin 2 in mammalian cells will be of value in evaluating various fusion proteins produced in mammalian cells for numerous applications.

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Molecular cloning of the chicken avidin cDNA

Cited by 73 publications

References 48 publications

Biochemical characterization and crystal structure of a recombinant hen avidin and its acidic mutant expressed in Escherichia coli

Biochemical characterization and crystal structure of a recombinant hen avidin and its acidic mutant expressed in Escherichia coli

Expression and purification of a recombinant avidin with a lowered isoelectric point in Pichia pastoris

High-level expression of tamavidin 2 in human cells by codon-usage optimization

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