Engineering domain fusion chimeras from I-OnuI family LAGLIDADG homing endonucleases.
Although engineered LAGLIDADG homing endonucleases (LHEs) are finding increasing applications in biotechnology, their generation remains a challenging, industrial-scale process. As new single-chain LAGLIDADG nuclease scaffolds are identified, however, an alternative paradigm is emerging: identification of an LHE scaffold whose native cleavage site is a close match to a desired target sequence, followed by small-scale engineering to modestly refine recognition specificity. The application of this paradigm could be accelerated if methods were available for fusing N- and C-terminal domains from newly identified LHEs into chimeric enzymes with hybrid cleavage sites. Here we have analyzed the structural requirements for fusion of domains extracted from six single-chain I-OnuI family LHEs, spanning 40-70% amino acid identity. Our analyses demonstrate that both the LAGLIDADG helical interface residues and the linker peptide composition have important effects on the stability and activity of chimeric enzymes. Using a simple domain fusion method in which linker peptide residues predicted to contact their respective domains are retained, and in which limited variation is introduced into the LAGLIDADG helix and nearby interface residues, catalytically active enzymes were recoverable for ≈ 70% of domain chimeras. This method will be useful for creating large numbers of chimeric LHEs for genome engineering applications.
Department of Immunology, University of Washington, Seattle, WA 98195, USA.
Sarah
S
Baxter
Abigail R
AR
Lambert
Ryan
R
Kuhar
Jordan
J
Jarjour
Nadia
N
Kulshina
Fabio
F
Parmeggiani
Patrick
P
Danaher
Jacob
J
Gano
David
D
Baker
Barry L
BL
Stoddard
Andrew M
AM
Scharenberg
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020029:Base Pairing; D004247:DNA / Q000737:chemistry; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D010455:Peptides; D011993:Recombinant Fusion Proteins; D004247:DNA; D004706:Endodeoxyribonucleases