53
the end of that gene, you can PCR amplify the second copy of
gene (with a stop codon) to add a suitable linker and directly
ligate this amplified gene at the 3′ end of the first gene copy
using the restriction site. When no restriction site is available,
the genes can be fused together by overlap PCR.
17. Linker connecting the original N- and C-termini: Linker
length and composition can interfere with the folding, topology,
and stability of the protein, so it must be rationally designed.
Here are some considerations. (a) Length—the distance
between the alpha carbons of the N- and C-termini residues
can be determined using Pymol or similar molecular visualization software. Depending on the secondary structure of the
linker residues, one amino acid typically spans a distance of
1.5–3.5 Å. Linkers that bridge distances greater than 10 Å
require more careful design. Depending on the situation, the
protein’s surface properties must be taken into account. (b)
Composition—Flexible backbones can help accommodate
emerging conformational strains; thus, in most cases, glycinerich (Gly) bridges are preferred. Typically, a GlySerGlyGly
linker is sufficient to span a distance of 7–8 Å. However, linkers
of more diverse composition can be tested.
18. Inter-domain linker: In order to alleviate possible disturbances
caused by domain insertion, we usually add two residues to the
new termini of the permuted protein. This can be done using
degenerate primers with two codons of 5′-NNK-3′ at 5′ end of
the forward and reverse primers. Note that the reverse primer
must encode the reverse complement of 5′-(NNK) 2 -3′.
19. Reducing library size: For large proteins, purchasing comprehensive sets of primers can be expensive. To decrease primer
costs and the library size, you can create select circular permutations that focus on residues that are solvent accessible, flexible,
loosely packed, and between secondary structure elements.
20. Phosphorylated primers can be ordered from most manufacturers or unphosphorylated primers can be phosphorylated
using T4 Polynucleotide Kinase (New England Biolabs,
Ipswich, MA) following the manufacturer’s instructions.
Alternatively, you can phosphorylate the PCR product.
21. For the best results, do not exceed 10 ng of vector per μL of the
ligation reaction. Note that the ligase used is high concentration ligase (2,000,000 units/mL) for this blunt end ligation
reaction.
22. The ligation reaction can be scaled up. For the best results,
prepare a master mix of the ligation and split it into 20 μL aliquots for the incubation.
23. Alternatively, you can elute in 6 μL of DNA but this may
decrease your yield.
Engineering Protein Switches by Domain Insertion
the end of that gene, you can PCR amplify the second copy of
gene (with a stop codon) to add a suitable linker and directly
ligate this amplified gene at the 3′ end of the first gene copy
using the restriction site. When no restriction site is available,
the genes can be fused together by overlap PCR.
17. Linker connecting the original N- and C-termini: Linker
length and composition can interfere with the folding, topology,
and stability of the protein, so it must be rationally designed.
Here are some considerations. (a) Length—the distance
between the alpha carbons of the N- and C-termini residues
can be determined using Pymol or similar molecular visualization software. Depending on the secondary structure of the
linker residues, one amino acid typically spans a distance of
1.5–3.5 Å. Linkers that bridge distances greater than 10 Å
require more careful design. Depending on the situation, the
protein’s surface properties must be taken into account. (b)
Composition—Flexible backbones can help accommodate
emerging conformational strains; thus, in most cases, glycinerich (Gly) bridges are preferred. Typically, a GlySerGlyGly
linker is sufficient to span a distance of 7–8 Å. However, linkers
of more diverse composition can be tested.
18. Inter-domain linker: In order to alleviate possible disturbances
caused by domain insertion, we usually add two residues to the
new termini of the permuted protein. This can be done using
degenerate primers with two codons of 5′-NNK-3′ at 5′ end of
the forward and reverse primers. Note that the reverse primer
must encode the reverse complement of 5′-(NNK) 2 -3′.
19. Reducing library size: For large proteins, purchasing comprehensive sets of primers can be expensive. To decrease primer
costs and the library size, you can create select circular permutations that focus on residues that are solvent accessible, flexible,
loosely packed, and between secondary structure elements.
20. Phosphorylated primers can be ordered from most manufacturers or unphosphorylated primers can be phosphorylated
using T4 Polynucleotide Kinase (New England Biolabs,
Ipswich, MA) following the manufacturer’s instructions.
Alternatively, you can phosphorylate the PCR product.
21. For the best results, do not exceed 10 ng of vector per μL of the
ligation reaction. Note that the ligase used is high concentration ligase (2,000,000 units/mL) for this blunt end ligation
reaction.
22. The ligation reaction can be scaled up. For the best results,
prepare a master mix of the ligation and split it into 20 μL aliquots for the incubation.
23. Alternatively, you can elute in 6 μL of DNA but this may
decrease your yield.
Engineering Protein Switches by Domain Insertion