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 E. coli strain inserts O-methyl-L-tyrosine into proteins that contain a UAG stop codon, resulting in a full-length target protein containing the modified amino acid. Had the mutant gene been expressed in wild-type E. coli, a truncated version of the protein would have been produced. This system may be manipulated to insert a variety of different amino acid analogues into specified sites within proteins in an effort to produce functional proteins with altered activities compared with the native form. In a similar approach to this problem, researchers modified a portion of the valine-tRNA synthetase gene so that the altered enzyme was able to add the nonstandard amino acid aminobutyrate to a specific tRNA for subsequent incorporation into proteins. While the full potential of these approaches has yet to be realized, it is nevertheless clear that it is now possible to produce proteins containing unusual chemical structures and possibly having unique properties.
Figure 3.49 Production of a protein with a modified (nonstandard amino acid) side chain. The start codon is highlighted in green, and the stop codons are in red. The inserted amino acid analogue is shown in blue.
Random Mutagenesis
In those cases where the amino acid changes that will result in the desired properties are unknown, a library of mutated sequences is generated by randomly altering individual nucleotides within a structural gene. Most of the mutations will decrease the functioning of the encoded protein, and therefore an efficient screening process is required to identify proteins with the rare mutations that result in beneficial changes.
Error-Prone PCR
Some of the temperature-stable DNA polymerases that are used to amplify target DNA by PCR occasionally insert incorrect nucleotides during DNA replication. If one is attempting to amplify a DNA with high fidelity, this is obviously a problem. On the other hand, if the construction of a library of mutants of the target gene is the objective, then this approach is a useful method for random mutagenesis. Error-prone PCR is performed using DNA polymerases that lack proofreading activity, such as Taq DNA polymerase. The error rate may be increased by increasing the concentration of Mg2+ to stabilize noncomplementary base pairs. Addition of Mn2+, and/or unequal amounts of the four deoxynucleoside triphosphates to the reaction buffer may also increase the error rate. The primer annealing sites on the template DNA define the region to be altered, and the number of nucleotide substitutions per template increases with the number of PCR cycles and the length of the template. Following error-prone PCR, the randomly mutagenized DNA is cloned into an expression vector and screened for altered or improved protein activity. The DNA from those clones that encode the desired activity is isolated and sequenced to determine the relevant changes to the target DNA.
Random Insertion/Deletion Mutagenesis
While error-prone PCR is quite commonly used to introduce random changes into a target gene, it is somewhat limited in the types of changes that can be introduced. Since errors are typically introduced into DNA at no more than one or two per 1,000 nucleotides, only single nucleotides are replaced within a triplet codon, yielding a limited number of amino acid changes from each mutated DNA molecule. As an alternative to error-prone PCR, researchers have developed the technique of random insertion/deletion mutagenesis. With this approach, it is possible to delete a small number of nucleotides at random positions along the gene and, at the same time, insert either specific or random sequences into that position. This method entails the following steps (Fig. 3.50).
1 An isolated gene fragment with different restriction endonuclease sites at each end is ligated at one end to a short nonphosphorylated adaptor that leaves a small gap in one strand of the DNA. The gap is a consequence of the fact that the 5′ nucleotide on the adaptor is not phosphorylated and therefore cannot be ligated to an adjacent 3′-OH group on the gene fragment.
2 After restriction enzyme digestion that creates compatible sticky ends, the gene fragment is recircularized with T4 DNA ligase to create a circular double-stranded gene fragment with a gap in one of the strands.
3 The gapped strand is degraded by digestion with the enzyme T4 DNA polymerase, which has exonuclease activity.
4 Each single-stranded DNA molecule is randomly cleaved at a single positions by treating it with a cerium(IV)–ethylenediaminetetraacetic acid (EDTA) complex.
5 The linear single-stranded DNA molecules are ligated at each end with adaptors that contain annealing sites for PCR primers, one of which contains several additional nucleotides selected for insertion. The entire mutagenesis library is amplified by PCR.
6 The adaptors are removed by restriction enzyme digestion and the constructs are made blunt ended by filling in the single-stranded overhangs using the Klenow fragment of E. coli DNA polymerase I before the DNA molecules are recircularized
