Molecular Biotechnology. Bernard R. Glick

enhanced characteristics can be created for therapeutic or industrial applications. For example, the catalytic efficiency, allosteric regulation, cofactor requirement, or substrate specificity of an enzyme may be improved. The latter may decrease undesirable side effects of a therapeutic enzyme. The thermal tolerance and/or pH stability of a protein may be increased, enabling the mutant protein to be used under conditions that would inactivate the native version. The sensitivity of a protein to cellular proteases may be decreased, which would increase the recoverable yield of the protein.

Directed Mutagenesis

      In some cases, it is possible to predict in advance which individual amino acids or short sequences of amino acids contribute to a particular physical, kinetic, or chemical property. The process for generating specific amino acid changes by changing the coding sequence at a targeted site in a gene is called site-directed mutagenesis. It is important to keep in mind that a particular property of a protein may be the consequence of two or more amino acids that are far apart from each other in the linear sequence but are juxtaposed as a result of the folding of the protein. In this case, two or more amino acids may have to be changed to produce a protein with the desired properties. Predicting which amino acids of a protein should be changed to attain a specific property generally requires that the three-dimensional structure of the protein, or a similar protein, has been well characterized by X-ray crystallographic analysis. However, for many proteins, such detailed information is often lacking, so site-directed mutagenesis becomes a trial-and-error strategy in which changes are made to those nucleotides that are most likely to yield a particular change in a protein property. Then, of course, the protein encoded by each mutated gene has to be tested to ascertain whether the mutagenesis process has indeed generated the desired change.

Site-Directed Mutagenesis by Overlap Extension PCR

      A straightforward site-directed mutagenesis protocol introduces defined nucleotide substitutions into a gene by overlap extension PCR. Two pairs of oligonucleotide primers are used in the PCR; one set of flanking primers anneals to the ends of the target gene (often a cloned gene), and the other set consists of overlapping, internal primers that carry the mutation. While the 3′ end of a primer must be perfectly complementary to the annealing site on the template DNA to prime DNA synthesis, mismatched nucleotides at the 5′ end do not affect the reaction. The target gene is initially amplified in two separate reactions, to generate overlapping (“left” and “right”) fragments (Fig. 3.47). In each of the two reactions, one of the PCR primers (an internal primer) carries the mutation and the other is a flanking primer. After PCR amplification, the products are purified and the left and right fragments are combined. Denaturation and reannealing of the mixed fragments produce some DNA molecules that hybridize in the overlapping, complementary, mutated sequences. DNA polymerase is added to extend the strands to form double-stranded DNA molecules. These molecules are amplified by PCR with the flanking primers to enrich for full-length DNA molecules. The amplified DNA is then cloned into a suitable plasmid vector; this is facilitated by inclusion of suitable restriction enzyme sites in the 5′ ends of the flanking primers. This procedure results in the production of an altered gene that has mutated sites in the region of the overlap of the internal oligonucleotides.

      Figure 3.47 Site-directed mutagenesis by overlap extension PCR. The left and right portions of the target DNA are amplified separately by PCR. The primers are shown by horizontal arrows. Primers that carry the mutation are depicted as a line with a spike; a spike denotes a position that contains a nucleotide that is not found in the native gene. The amplified fragments are purified, denatured to make them single stranded, and then reannealed. Regions of overlap are formed between complementary mutation-producing sequences. The single-stranded regions are made double-stranded with DNA polymerase, and then the entire fragment is amplified by PCR. The resultant product is digested with restriction endonucleases A and B and then cloned into a vector that has been digested with the same enzymes.

Site-Directed Mutagenesis by Inverse PCR

      Nucleotide substitutions, deletions, or insertions can be introduced into a target gene that has been cloned into a plasmid in a procedure known as inverse PCR. In this case, the entire plasmid is amplified, which restricts the size of the plasmid to less than about 10 kb. The oligonucleotide primers used in the inverse PCR anneal to adjacent sequences in the target gene but are divergently oriented, that is, their 3′ ends are directed away from each other. For point mutations, nucleotide changes are introduced in the middle of one of the primers (Fig. 3.48). To create deletion mutations, primers must flank the region of target DNA to be deleted and be perfectly matched to their annealing sites (Fig. 3.48). To create mutations with long insertions, a stretch of mismatched nucleotides is added to the 5′ end of one or both primers, while for mutations with short insertions, a stretch of nucleotides is added in the middle of one of the primers (Fig. 3.48). PCR amplification yields linear double-stranded DNA products that are circularized by ligation with T4 DNA ligase. Ligation requires that the 5′ ends of the linear DNA molecules are phosphorylated and therefore either the primers must be phosphorylated or the PCR products must be phosphorylated using the enzyme polynucleotide kinase. Finally, the recircularized plasmid DNA is transformed into E. coli by any standard procedure. Since this protocol yields a very high frequency of plasmids with the desired mutation, screening three or four clones by sequencing the target DNA is usually sufficient to find the desired mutation. Given its simplicity and effectiveness, this procedure has come to be widely used to introduce a specified point mutation, insertion, or deletion into a cloned gene.

      Figure 3.48 Overview of the basic methodology to introduce point mutations, insertions, or deletions into DNA cloned into a plasmid. The forward and reverse primers are shown in red and green, respectively. The solid circles represent template DNA. The dotted lines represent newly synthesized DNA. The X indicates an altered nucleotide(s).

Mutant Proteins with Unusual Amino Acids

      Essentially any protein can be altered by substituting one amino acid for another using site-directed mutagenesis. However, this approach is limited to the 20 amino acids that are normally used in protein synthesis. One way to increase the diversity of the proteins formed after mutagenesis is to introduce synthetic amino acids with unique side chains at specific sites. To do this, E. coli was engineered to produce both a novel transfer RNA (tRNA) that is not recognized by any of the existing E. coli aminoacyl-tRNA synthetases but nevertheless functions in translation and a new aminoacyl-tRNA synthetase that aminoacylates only that novel tRNA. A novel tRNA and unique aminoacyl-tRNA synthetase pair from the archaebacterium Methanococcus jannaschii was used as a starting point for this system. The tyrosine-tRNA synthetase from M. jannaschii can add an amino acid to an amber suppressor tRNA that is a mutant form of tyrosine-tRNA. An amber suppressor tRNA is a modified tRNA that can insert an amino acid into a protein in places where the mRNA contains an amber codon, UAG, which normally is a stop codon that directs the cessation of protein synthesis. To prevent the translational fusion of proteins whose mRNAs normally contain a UAG stop codon with downstream proteins, in vivo suppression is always less than 100% and is often dependent upon the nucleotides surrounding the stop codon. The amino acid specificity of the tyrosine-tRNA synthetase from M. jannaschii is altered by random mutagenesis of its gene so that, instead of tyrosine, it catalyzes the addition of O-methyl-L-tyrosine onto the tRNA. A cloned version of the target gene is modified by site-directed mutagenesis so that it contains a 5′-TAG-3′ in that portion of the DNA that encodes the amino acid that is targeted for change to O-methyl-l-tyrosine (Fig. 3.49). Once the modified DNA has been created, it is used to transform an E. coli strain that was previously engineered to produce the O-methyl-L-tyrosine-tRNA. The engineered