Recombinant NeutraLite Avidin: a non-glycosylated, acidic mutant of chicken avidin that exhibits high affinity for biotin and low non-specific binding properties

• Published in: FEBS letters 2000-02, Vol.467 (1), p.31-36
• Main Authors: Marttila, Ari T., Laitinen, Olli H., Airenne, Kari J., Kulik, Tikva, Bayer, Edward A., Wilchek, Meir, Kulomaa, Markku S.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acid Substitution - genetics; Animals; Avidin - chemistry; Avidin - genetics; Avidin - isolation & purification; Avidin - metabolism; Avidin-biotin technology; Baculoviridae - genetics; Baculoviridae - metabolism; Biotin - analogs & derivatives; Biotin - metabolism; Cells, Cultured; Chick Embryo; DNA - metabolism; Endopeptidase K - metabolism; Glycosylation; Humans; Isoelectric Point; Kinetics; Molecular Sequence Data; Mutation - genetics; Non-glycosylated mutant; Non-specific binding; Protein Binding; Protein Engineering; Protein Structure, Quaternary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Thermodynamics
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(00)01119-4

A recombinant non-glycosylated and acidic form of avidin was designed and expressed in soluble form in baculovirus-infected insect cells. The mutations were based on the same principles that guided the design of the chemically and enzymatically modified avidin derivative, known as NeutraLite Avidin. In this novel recombinant avidin derivative, five out of the eight arginine residues were replaced with neutral amino acids, and two of the lysine residues were replaced by glutamic acid. In addition, the carbohydrate-bearing asparagine-17 residue was altered to an isoleucine, according to the known sequences of avidin-related genes. The resultant mutant protein, termed recombinant NeutraLite Avidin, exhibited superior properties compared to those of avidin, streptavidin and the conventional NeutraLite Avidin, prepared by chemo-enzymatic means. In this context, the recombinant mutant is a single molecular species, which possesses strong biotin-binding characteristics. Due to its acidic p I, it is relatively free from non-specific binding to DNA and cells. The recombinant NeutraLite Avidin retains seven lysines per subunit, which are available for further conjugation and derivatization.