35
structural defect is introduced into the POI which is then “swapped
out” by fragment binding. A reliable tuning mutation site consists
of a large, buried hydrophobic residue (Leu, Ile, Phe, or Tyr) that
is in the duplicated portion of the POI and packs against hydrophobic residues in the unduplicated portion (Fig. 2b). A single
tuning site should (and usually can) be selected that will service all
fragment lengths to be tested. Tuning consists of substituting
progressively smaller hydrophobic residues (Val, Ala, Gly, and for
extreme destabilization, a charged residue) into that site to progressively destabilize the POI.
4 Step 2 of AFF Protocol: Gene Construction
AFF genes are generated by constructing two half-genes corresponding to the N- and N′-frames, mutating them as needed, and
then fusing them together. Keeping the frames separate is necessary because mutagenic PCR primers will bind to two locations in
the full-length AFF gene due to sequence duplication. This redundancy also makes it difficult to obtain fully synthetic AFF genes
from commercial sources.
A His 6–8 sequence or other purification/expression tag can be
introduced at this point. The tag should be expressed at the terminus
of the protein that comprises the N′-fold, i.e., at the N-terminus of
POI-AFF if a C-terminal fragment was duplicated, or at the
C-terminus if an N-terminal fragment was duplicated. This arrangement will help select against purifying degradation products,
because if a cellular protease cleaves POI-AFF it will likely do so by
attacking the less stable N′-fold. The following steps describe
construction of an AFF-POI in which a C-terminal fragment is
duplicated and appended to the N-terminus; analogous steps are
taken for an AFF-POI with a duplicated N-terminal fragment.
Introduce binding, Cys, and tuning mutations into the WT POI
gene as dictated in Subheading 3. Amplify the N-frame half-gene
by annealing primer 1 (containing a stop codon) to the 3′-end of
the above-modified POI gene, and primer 2 to the 5′-end
(Fig. 4a). Primer 2 encodes for the C-terminal half of the desired
linker and bears a restriction site of choice at its 5′-end. The restriction site necessarily becomes incorporated into the linker and for
this reason we favor the Not I restriction site because it encodes for
Ala-Ala-Ala.
Introduce an N′-frame tuning mutation into the WT POI gene if
necessary. Design primer 3 such that it binds the POI gene at a
position that defines the start of the duplicate polypeptide segment
(and the N-terminus of POI-AFF) (Fig. 4a). Primer 3 begins with
a codon for Cys. Primer 4 anneals to the 3′-end of the POI gene,
4.1 N-frame
Half-Gene
4.2 N′-frame
Half-Gene
Engineering Allosteric Protein Switches
structural defect is introduced into the POI which is then “swapped
out” by fragment binding. A reliable tuning mutation site consists
of a large, buried hydrophobic residue (Leu, Ile, Phe, or Tyr) that
is in the duplicated portion of the POI and packs against hydrophobic residues in the unduplicated portion (Fig. 2b). A single
tuning site should (and usually can) be selected that will service all
fragment lengths to be tested. Tuning consists of substituting
progressively smaller hydrophobic residues (Val, Ala, Gly, and for
extreme destabilization, a charged residue) into that site to progressively destabilize the POI.
4 Step 2 of AFF Protocol: Gene Construction
AFF genes are generated by constructing two half-genes corresponding to the N- and N′-frames, mutating them as needed, and
then fusing them together. Keeping the frames separate is necessary because mutagenic PCR primers will bind to two locations in
the full-length AFF gene due to sequence duplication. This redundancy also makes it difficult to obtain fully synthetic AFF genes
from commercial sources.
A His 6–8 sequence or other purification/expression tag can be
introduced at this point. The tag should be expressed at the terminus
of the protein that comprises the N′-fold, i.e., at the N-terminus of
POI-AFF if a C-terminal fragment was duplicated, or at the
C-terminus if an N-terminal fragment was duplicated. This arrangement will help select against purifying degradation products,
because if a cellular protease cleaves POI-AFF it will likely do so by
attacking the less stable N′-fold. The following steps describe
construction of an AFF-POI in which a C-terminal fragment is
duplicated and appended to the N-terminus; analogous steps are
taken for an AFF-POI with a duplicated N-terminal fragment.
Introduce binding, Cys, and tuning mutations into the WT POI
gene as dictated in Subheading 3. Amplify the N-frame half-gene
by annealing primer 1 (containing a stop codon) to the 3′-end of
the above-modified POI gene, and primer 2 to the 5′-end
(Fig. 4a). Primer 2 encodes for the C-terminal half of the desired
linker and bears a restriction site of choice at its 5′-end. The restriction site necessarily becomes incorporated into the linker and for
this reason we favor the Not I restriction site because it encodes for
Ala-Ala-Ala.
Introduce an N′-frame tuning mutation into the WT POI gene if
necessary. Design primer 3 such that it binds the POI gene at a
position that defines the start of the duplicate polypeptide segment
(and the N-terminus of POI-AFF) (Fig. 4a). Primer 3 begins with
a codon for Cys. Primer 4 anneals to the 3′-end of the POI gene,
4.1 N-frame
Half-Gene
4.2 N′-frame
Half-Gene
Engineering Allosteric Protein Switches