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Directed Evolution of Arginine Kinase Oligomerization
Author(s) -
Swegal Warren,
Fraga Dean,
Edmiston Paul L
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1004.2
Subject(s) - mutagenesis , arginine kinase , dimer , kinase , dna shuffling , mutation , mutant , protein kinase domain , biochemistry , biology , chemistry , microbiology and biotechnology , directed evolution , fusion protein , protein kinase a , function (biology) , genetics , gene , arginine , amino acid , recombinant dna , organic chemistry
A new technique for directed evolution of protein oligomerization was developed. The system is based on fusing a target protein to the DNA binding domain of a cI lambda phage inhibitor protein lacking an oligomerization domain required for function (Mariño‐Ramírez, et al. J. Bact . 186:1311). Activation of the λ inhibitor can only proceed if the target undergoes mutational events that lead to dimerization, thus providing a selectable means for protein oligomerization. The phosphogen kinase (PK) family was used as a model system to test the technique. Within the PK family, arginine kinase (AK) is a monomer, yet the other prominent member, creatine kinase, is a homodimer. The two share a high degree of tertiary structure similarity making them a target for directed evolution to create a dimer from monomeric AK. Prior to random mutagenesis and screening, a cloned variant of L. polyphemus AK that possessed ten site‐directed mutations was created to incorporate key residues found within the dimer interface of CK, however these mutations alone do not produce a dimeric AK. Ethylenenitrosourea was used to randomly mutate the AK gene in vivo . After mutation steps, cells containing the fusion were exposed to λKH5‐h80 with increasing levels of stringency by adding variable levels of ITPG as a second means of inhibitor activation. The AKs of surviving clones were sequenced to determine the number and identity of the mutational events.