289
end, we established the PATCHY (primer-aided truncation for the
creation of hybrid proteins) method for the efficient generation of
hybrid gene libraries with defined composition [10].
Prior to this, alternate strategies for creating hybrid gene libraries had been proposed and successfully applied for the construction
of novel signal receptors, in particular the methods SHIPREC
(sequence homology-independent protein recombination) and
ITCHY (incremental truncation for the creation of hybrid proteins) [11–14]. However, for fragmentation of the parental genes,
these methods rely on endonucleolytic or exonucleolytic DNA
cleavage, both of which are challenging to titrate. Moreover, it is
inherently difficult to confine hybrid fusions to specific regions of
the parental genes such as their linker regions. Taken together,
these limitations have hampered the wider application of either
method [15]. In contrast, PATCHY circumvents problems arising
from nucleolytic DNA fragmentation by means of PCR amplification with sets of staggered primers to truncate the parental genes
and thus produce linker libraries of defined composition (Fig. 2).
As detailed in this chapter (see Subheading 3.1), PATCHY starts
from a template construct in which two parental gene fragments
are concatenated such that they are separated by a nucleotide
stretch that introduces a frameshift and a unique restriction site.
The template is then amplified in a one-pot PCR reaction with sets
of forward and reverse primers. Both primer sets are staggered in
LOV
STA T T S
L
Y
L
F2
PAS
HisK
L
F1
PAS
BsYtvA
BjFixL
YF1
1
124 147
261
1
128 143
255 282
505
HisK
LOV
L
F2
linker length i BsYtvA
linker length j BjFixL
a
b
c
Fig. 1 (a) Domain architecture of BsYtvA, BjFixL, and YF1. The hybrid YF1 consists of the BsYtvA LOV domain
fused to the BjFixL histidine kinase, where almost the entire linker derives from BjFixL. (b) The crystal structure
(PDB entry 4GCZ [9]) of homodimeric YF1 in its dark-adapted state shows the coiled-coil linker between the
LOV photosensor and histidine kinase effector modules. (c) Schematic of the possible linker combinations for
hybrids between the BsYtvA photosensor and the BjFixL effector modules
Generation of Hybrid Gene Libraries
end, we established the PATCHY (primer-aided truncation for the
creation of hybrid proteins) method for the efficient generation of
hybrid gene libraries with defined composition [10].
Prior to this, alternate strategies for creating hybrid gene libraries had been proposed and successfully applied for the construction
of novel signal receptors, in particular the methods SHIPREC
(sequence homology-independent protein recombination) and
ITCHY (incremental truncation for the creation of hybrid proteins) [11–14]. However, for fragmentation of the parental genes,
these methods rely on endonucleolytic or exonucleolytic DNA
cleavage, both of which are challenging to titrate. Moreover, it is
inherently difficult to confine hybrid fusions to specific regions of
the parental genes such as their linker regions. Taken together,
these limitations have hampered the wider application of either
method [15]. In contrast, PATCHY circumvents problems arising
from nucleolytic DNA fragmentation by means of PCR amplification with sets of staggered primers to truncate the parental genes
and thus produce linker libraries of defined composition (Fig. 2).
As detailed in this chapter (see Subheading 3.1), PATCHY starts
from a template construct in which two parental gene fragments
are concatenated such that they are separated by a nucleotide
stretch that introduces a frameshift and a unique restriction site.
The template is then amplified in a one-pot PCR reaction with sets
of forward and reverse primers. Both primer sets are staggered in
LOV
STA T T S
L
Y
L
F2
PAS
HisK
L
F1
PAS
BsYtvA
BjFixL
YF1
1
124 147
261
1
128 143
255 282
505
HisK
LOV
L
F2
linker length i BsYtvA
linker length j BjFixL
a
b
c
Fig. 1 (a) Domain architecture of BsYtvA, BjFixL, and YF1. The hybrid YF1 consists of the BsYtvA LOV domain
fused to the BjFixL histidine kinase, where almost the entire linker derives from BjFixL. (b) The crystal structure
(PDB entry 4GCZ [9]) of homodimeric YF1 in its dark-adapted state shows the coiled-coil linker between the
LOV photosensor and histidine kinase effector modules. (c) Schematic of the possible linker combinations for
hybrids between the BsYtvA photosensor and the BjFixL effector modules
Generation of Hybrid Gene Libraries