Production of Biologically Active Recombinant Avidin in Baculovirus-Infected Insect Cells

Airenne, Kari J.; Oker‐Blom, Christian; Marjomäki, Varpu; Bayer, Edward A.; Wilchek, Meir; Kulomaa, Markku S.

doi:10.1006/prep.1996.0660

Cited by 55 publications

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“…Recombinant avidin has also been recently obtained in a baculovirus expression system, obtaining a glycosylated protein with properties analogous to those of natural avidin, including the high thermo-resistance [13]. Similarly, both recombinant and [E3,E9,D26,L124]avidin saturated with biotin resisted heating at 80°C in the presence of 1% SDS (not shown), indicating that glycosylation does not contribute to avidin physical stability.…”

Section: Discussionmentioning

confidence: 98%

“…Avidin has been expressed in Escherichia coli where it is accumulated in inclusion bodies, with only a minor proportion of soluble and functional protein [3,11]. Correctly assembled and functional recombinant avidin has been obtained from transfected mammalian cell lines [12] and, more recently, in a baculovirus expression system [13]. Mutated avidins, however, have not been described so far.…”

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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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Section: Discussionmentioning

confidence: 98%

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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“…The primary source for the commercial production of avidin is the extraction and purification from chicken egg white. More recently, biologically active recombinant isoforms have been produced in several expression systems, including Escherichia coli [11,12], Baculovirus-infected cells [13] and maize [14].…”

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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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“…The virus vectors for producing the AVR proteins were constructed and amplified according to the Bac-To-Bac system instructions. Recombinant AVR proteins were produced in Spodoptera frugiperda (Sf 9) insect cells as previously reported [22]. Proteins were purified from the cells using affinity chromatography on a 2-iminobiotin (AVR1, AVR3, AVR4\5-7) and\or biotinagarose column (AVR1 and AVR2) as previously described [18].…”

Section: Production and Purification Of Recombinant Avr Proteinsmentioning

confidence: 99%

“…However, all AVRs showed different patterns on SDS\PAGE as compared with avidin [22] (Figure 4). One-step 2-iminobiotin (AVR1, AVRs 3-7) -and\or biotin (AVRs 1 and 2) -agarose affinity chromatography yielded highly homogenous proteins showing no contaminants as judged by SDS\PAGE analysis.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Chicken avidin-related proteins show altered biotin-binding and physico-chemical properties as compared with avidin

LAITINEN

Hytönen

Ahlroth

et al. 2002

Biochemical Journal

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Chicken avidin and bacterial streptavidin are proteins familiar from their use in various (strept)avidin—biotin technological applications. Avidin binds the vitamin biotin with the highest affinity known for non-covalent interactions found in nature. The gene encoding avidin (AVD) has homologues in chicken, named avidin-related genes (AVRs). In the present study we used the AVR genes to produce recombinant AVR proteins (AVRs 1, 2, 3, 4/5, 6 and 7) in insect cell cultures and characterized their biotin-binding affinity and biochemical properties. Amino acid sequence analysis and molecular modelling were also used to predict and explain the properties of the AVRs. We found that the AVR proteins are very similar to avidin, both structurally and functionally. Despite the numerous amino acid substitutions in the subunit interface regions, the AVRs form extremely stable tetramers similar to those of avidin. Differences were found in some physico-chemical properties of the AVRs as compared with avidin, including lowered pI, increased glycosylation and, most notably, reversible biotin binding for two AVRs (AVR1 and AVR2). Molecular modelling showed how the replacement Lys111→isoleucine in AVR2 alters the shape of the biotin-binding pocket and thus results in reversible binding. Both modelling and biochemical analyses showed that disulphide bonds can form and link monomers in AVR4/5, a property not found in avidin. These, together with the other properties of the AVRs described in the present paper, may offer advantages over avidin and streptavidin, making the AVRs applicable for improved avidin—biotin technological applications.

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Production of Biologically Active Recombinant Avidin in Baculovirus-Infected Insect Cells

Cited by 55 publications

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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

Chicken avidin-related proteins show altered biotin-binding and physico-chemical properties as compared with avidin

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