Engineering of chicken avidin: a progressive series of reduced charge mutants

Abstract

Avidin, a positively charged egg-white glycoprotein, is a widely used tool in biotechnological applications because of its ability to bind biotin strongly. The high pI of avidin (V10.5), however, is a hindrance in certain applications due to non-specific (charge-related) binding. Here we report a construction of a series of avidin charge mutants with pIs ranging from 9.4 to 4.7. Rational design of the avidin mutants was based on known crystallographic data together with comparative sequence alignment of avidin, streptavidin and a set of avidin-related genes which occur in the chicken genome. All charge mutants retained the ability to bind biotin tightly according to optical biosensor interaction analysis. In most cases, their thermal stability characteristics were indistinguishable from those of the wildtype avidin. Our results demonstrate that the charge properties of avidin can be modified without disturbing the crucial biotinbinding activity.z 1998 Federation of European Biochemical Societies.

Construction of a Dual Chain Pseudotetrameric Chicken Avidin by Combining Two Circularly Permuted Avidins

“…Biotin-Binding Assays-An IAsys biosensor was used to determine the affinity of the proteins toward 2-iminobiotin and the reversibility of biotin binding, essentially as previously described in detail (10). In the reversibility assay, the sample proteins were allowed to bind to a biotinylated cuvette surface in phosphate-buffered saline containing 1 M NaCl.…”

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

“…The amino acid sequence of an avidin with an isoelectric point of 7.2, which contained the K3E, K9D, R122A, and R124A mutations [16], was back-translated selecting the preferred codons in P. pastoris [23,24] (http:// www.kazusa.or.jp/codon). A non-glycosylated variant of this avidin was produced first.…”

“…This suggests that either the protein fold imposed by the sequence is not suitable to recognition and proteolytic cleavage by STE13 or that the amount of the protein produced exceeds the catalytic capacity of STE13 protease, preventing the effective cleavage of the fusion protein formed by the a-factor signal and the recombinant avidin [38][39][40]. Considering this N-terminal modification in combination with the primary sequence containing the K3E, K9D, R122A, and R124A mutations [16], the isoelectric point of the recombinant avidin is estimated at pI ¼ 5.4 [41].…”

“…However, the high-isoelectric point of avidin (pI ¼ 10.5) limits its field of applications. The production of either genetically [16,17] or chemically [18] modified avidin allows one to overcome this problem. For example, the existence of monomeric isoforms [19] makes avidin an ideal partner for the expression of fusion proteins.…”

“…A non-glycosylated acidic mutant is particularly useful in affinity-based drug targeting [17]. Moreover, avidin clones with a neutral or slightly acidic isoelectric point were reported to be expressed at higher levels in different expression systems [16,20].…”