Molecular Biotechnology. Bernard R. Glick

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 by T4 DNA ligase.

      7 The products are digested with appropriate restriction enzymes that flank the protein coding sequences and the mutated sequences are cloned into a plasmid vector to test for activity.

      Figure 3.50 A random insertion protocol to introduce random mutations into a gene of interest. The inserted DNA is shown in yellow. Adapted from Murakami et al., Nat. Biotechnol. 20:76–81, 2002.

      With this approach, it is possible to insert any small DNA fragment (carried on an adaptor) into the randomly cleaved single-stranded DNA, with the result that genes with a much greater number of modified nucleotides may be generated than by error-prone PCR. The mutations that are developed by this procedure may be used to select protein variants with a wide range of activities.

Random Mutagenesis with Degenerate Oligonucleotide Primers

      In addition to introducing a specific nucleotide substitution into a gene, overlap extension PCR can be used to incorporate any of the four nucleotides at defined positions to generate all the possible amino acid changes in a particular region of a protein. This pattern of sequence degeneracy is achieved by programming an automated DNA synthesis reaction to add a low level (usually a few percent) of each of the three alternative nucleotides each time a particular nucleotide is added during the synthesis of an oligonucleotide primer (Fig. 3.51). In this way, the oligonucleotide primer preparation contains a heterogeneous set of DNA sequences that will generate a series of mutations that are clustered in a defined portion of the target gene. The degenerate oligonucleotides are employed as “internal” PCR primers to amplify the left and right portions of the target gene in separate reactions (Fig. 3.47). Mixing, denaturing, and annealing the left and right fragments produces some DNA molecules that overlap by complementarity and can be extended by DNA polymerase to produce a library of altered genes that have mutated sites in the region of the overlap of the degenerate oligonucleotides.

      Figure 3.51 Chemical synthesis of oligonucleotide primers with any of the four nucleotides at defined positions. In this case, the flask with G phosphoramidite consists of a mixture of nucleotides, such as 94% G, 2% A, 2% C, and 2% T, leading to a mixture of oligonucleotides that may have A, C, or T at the sites where G is the specified nucleotide.

      Mutagenesis using degenerate oligonucleotides confers two advantages over targeted mutagenesis: (1) Detailed information regarding the roles of particular amino acids in the functioning of the protein is not required; (2) Unexpected mutants encoding proteins with a range of interesting and useful properties may be generated because the introduced changes are not limited to one amino acid. Of course, should none of the mutants yield a protein with the properties that are being sought, then it may be necessary to repeat the entire procedure with a set of degenerate primers that is complementary to a different region of the gene.

DNA