Enhanced Metal-Affinity Partitoning of Genetically Engineered Hirudin Variants in Polyethylene Glycol/Dextran Two/Phase Systems

概要

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Hirudin variants were constructed to exhibit an increased metal-binding affinity in an attempt to apply a metal-affinity partitioning process in a primary separation step for purification of hirudin. The hirudin variants were genetically engineered to contain additional surface-accessible histidines and produced by recombinant Saccharomyces cerevisitae. The partitioning behavior of these variants was compared with that of the wild type with a single surface-accessible histidine at position 51. Upon the addition of a small amount of Cu(II)IDA-PEG (Cu(III)iminodiacetic acid-polyethylene glycol) ligand to PEG/dextran two-phase systems, the hirudin variants with two or three surface-accessible histidines were more selectively partitioned into the PEG-rich phase than the wild type. Integrating protein engineering to metal-affinity partitioning offers the potential for general application of this technique to facilitate protein isolation, but the genetically engineered protein variants should be carefully constructed in a manner to minimize reduction of native protein acitivity.
社団法人日本生物工学会の論文
1994-01-25