Alanine-stretch scanning mutagenesis: a simple and efficient method to probe
protein structure and function
Alanine-stretch scanning mutagenesis: a simple and efficient method to probe protein structure and function
Fabrice
Lefèvre
1
,
Marie-Hélène
Rémy
1
and
Jean-Michel
Masson
1,2,
*
1
Institut de Pharmacologie et Biologie Structurale and
2
Institut National des Sciences Appliquées, 205 Route de Narbonne, F-31077
Toulouse
Cédex,
France
Received September 5, 1996;
Revised and Accepted November 19, 1996
ABSTRACT
We have developed a foolproof method to substitute a stretch of residues by
alanines. After the introduction of a
Pst
I site by IPCR, thus creating two alanine codons, additional codons are
introduced at this site through the use of an `alanine-stretch cartridge'. These cartridges comprise an antibiotic resistance
gene flanked on both sides by alanine codons. Excision of the resistance gene
by
Pvu
II then yields the correct insertion of codons. The method is both highly
reliable and flexible and should be of general use.
Alanine-scanning mutagenesis is a simple and widely used technique in the
determination of the catalytic or functional role of protein residues. Alanine
is the substitution residue of choice since it eliminates the side chain beyond
the [beta] carbon and yet does not alter the main-chain conformation (as can glycine or proline) nor does it impose
extreme electrostatic or steric effects (
1
). Nonetheless, in case of systematic analysis of a protein, single amino
replacements are quite laborious, so that direct substitution of a stretch of
residues by alanines, or insertion of alanines become more effective. In order
to avoid the drawbacks of the standard linker mutagenesis (cost of the
oligonucleotides, low yield of insertion, no direct selection for linker
insertions) we have developed an antibiotic resistance gene cartridge method
which enables a foolproof phenotypic selection for the insertion of the
alanines. Although any such mutagenesis could directly be performed by IPCR,
the use of antibiotic cartridges has several advantages. It is more cost
effective in case of systematic scanning. Our method is also perfectly
foolproof whereas IPCR often yields incorrect insertions when used with very
repetitive sequences such as alanine stretch sequences. Finally, the use of
alanine stretch cartridges allows progressive insertion, as shown in the
example below.
The outline of the method is as follows (Fig.
1
). Site-directed mutagenesis is first used to introduce a
Pst
I restriction site in the sequence of the gene of interest. The IPCR mutagenesis
method was chosen for its simplicity and high efficiency (
2
). (It should be noted that the
Pst
I and
Pvu
II sites of the vector bearing the cloned gene have to be eliminated beforehand,
since the method relies on the use of these sites). In the substitution
approach, this site is introduced so that the first codon of the five
substituted ones is replaced by the GCT codon, and that the last one is
replaced by GCA. The first base of the sixth codon is substituted by a guanine
in order to create a complete
Pst
I site. In the insertion approach, the GCTGCA sequence is introduced between two
codons, and the first base of the second codon is changed to a guanine.
REFERENCES
1 Cunningham,B.C. and Wells,J.A. (1989) Science, 244, 1081-1085.MEDLINE Abstract
2 Weiner,M.P., Costa L.G., Schoettlin,W., Cline,J., Marthur,E. and Bauer, J.C. (1994) Gene, 151, 119-123.MEDLINE Abstract
