Biochemistry and Molecular Biology - Belyasova N.A. 2002

Fundamentals of Genetic Engineering
Practical Application of Genetic Engineering Methods
Localized Mutagenesis and Protein Engineering

Introduction/32.html">Genetic Engineering techniques, particularly the cloning of individual genes or their parts and DNA Sequencing, have significantly refined mutagenesis methodology by overcoming the major limitations of classical approaches to inducing Mutations in genomes. Classical genetic analysis involves exposing an entire genome to a mutagenic agent in vivo, which induces random and frequently multiple mutations, severely complicating mutant identification. Mutant individuals are typically detected through altered phenotypic traits, whereas The Nature of the mutation can be determined following DNA sequencing. Modern Site-Directed Mutagenesis essentially operates in reverse: the Gene or segment of interest is first cloned, its Structure is determined via sequencing, and the desired modifications are then introduced in vitro. The consequences of the induced mutation are subsequently evaluated after introducing the mutant gene back into the host Organism.

The simplest approach to targeted mutagenesis involves treating a cloned DNA fragment with a mutagenic agent; however, this approach similarly results in random structural alterations within the fragment. More reliable and frequently employed Methods of targeted mutagenesis are performed without The Use of mutagens. The predominant Selection/21.html">Types of mutations generated are deletions, insertions, and nucleotide substitutions.

Deletions. These types of mutations are generated during targeted mutagenesis using endonucleases, which include both restriction and non-specific endonucleases. The simplest Application of Restriction Enzymes involves digesting a given genome with a restriction endonuclease that generates multiple double-stranded breaks with cohesive ends. The resulting fragments are then religated using DNA ligase, which can yield molecules lacking a specific DNA segment. This approach generates large deletions and is typically used in preliminary experiments to determine the Functions of relatively large regions of cloned DNA.

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Fig. 21.1. Generation of deletions in cloned DNA fragments using restriction enzymes. Foreign DNA is shown in dark, and vector DNA in light

Small deletions are produced as follows. The cloned fragment within a vector is cleaved at an appropriate site using a restriction enzyme (Fig. 21.1). The resulting linear molecule is treated with exonuclease III, which hydrolyzes one strand of the DNA starting from the 3'-end. This yields a set of molecules with single-stranded 5'- overhangs of varying lengths. These overhangs are subsequently digested with S1 nuclease, which is specific for single-stranded DNA, thereby forming deletions within the DNA molecule. Alternatively, Bal 31 exonuclease can be used, as it catalyzes the degradation of both strands from the ends of linear DNA molecules. The enzymatic reactions are controlled by adjusting the incubation time, Temperature, and Enzyme Concentration to induce deletions of varying lengths. Before circularization, the resulting deletion variants of linear DNA are frequently outfitted with linkers to ensure restriction sites are present near the deletion site. Various other modifications of these described methods also exist.

Insertions. To generate insertions, cloned DNA is cleaved with a restriction enzyme or a non-specific endonuclease, followed by ligation of the resulting fragments in the presence of the segment intended for insertion. Chemically synthesized polylinkers (Chapter 20) are most commonly used as such segments.

Insertions, much like deletions, can disrupt the integrity of a gene or The structure of its regulatory regions, leading to the synthesis of a defective protein (typically inactive in the case of large deletions or frameshifts) or altering the METABOLISM/31.html">Transcription process of the gene of interest. This approach is most frequently employed to generate regulatory mutants and construct expression vectors (Chapter 20).

Point mutations. These mutations involve nucleotide substitutions and can be generated through several approaches, including cytosine deamination, incorporation of nucleotide analogues, and misincorporation of NUCLEOTIDES during gap repair, among others.

The first approach relies on the fact that cytosine residues in single-stranded DNA can be deaminated to uracil via bisulfite ion Treatment. Single-stranded regions in DNA are typically generated near restriction sites, for instance, through the action of exonuclease III. Following bisulfite treatment, the single-stranded gaps are filled in using DNA polymerase, and the ends are ligated. At sites where uridylate was formed instead of cytidylate during deamination, adenylate will occupy the complementary position, resulting in the replacement of a GC base pair with an AT pair during the Replication of such a molecule.

Another strategy for inducing substitutions involves treating cloned DNA with a restriction endonuclease in the presence of ethidium bromide, which intercalates between base-pair planes and disrupts the duplex structure. This causes a single-strand break (nick) in the DNA. A small gap is created at the nick site and subsequently filled in using DNA polymerase in the presence of dATP, dGTP, dCTP, and N-4-hydroxycytosine triphosphate instead of dTTP. Hydroxycytosine triphosphate is incorporated into the chain in place of thymidylate, but during DNA replication, it pairs equally well with both adenylate and guanylate. Consequently, following an additional round of replication, the incorporation of guanylate leads to an AT→GC substitution at this site (Fig. 21.2). Because this method introduces nucleotide substitutions within a restriction site, vectors with the original sequence can be easily distinguished from mutant ones simply by treating them with the restriction enzyme used in the experiment: mutant molecules will remain uncleaved.

Fig. 21.2. Introduction of nucleotide substitutions during site-directed mutagenesis

A similar method relies on using only three of the four possible nucleotides when filling in a single-stranded gap with DNA polymerase. In most cases, the enzyme stalls at the position in the molecule where the nucleotide complementary to the missing one is encountered. Occasionally, however, DNA polymerase makes an error and incorporates one of the three available nucleotides. This leads to The formation of circular molecules containing unpaired, non-complementary nitrogenous bases. When these vectors are introduced into bacterial Cells, this damage is partially repaired in a fraction of the molecules. As a result, the original sequence is restored in half of the molecules following replication, whereas the mutation becomes fixed in the other half. Mutant molecules can be identified using the aforementioned approach.

Site-specific mutagenesis. The localized mutagenesis methods described above are characterized by the fact that the mutation sites are chosen at random. In contrast, site-specific mutagenesis techniques allow mutations to be introduced into a precisely defined region of a gene. This is achieved using chemically synthesized oligonucleotides with a predetermined sequence. The method is advantageous because it does not require convenient restriction sites to be present. The technique is based on the formation of heteroduplexes between a synthetic oligonucleotide containing the mutation and a complementary single-stranded vector DNA.

The Procedure is carried out as follows. A short oligonucleotide (8–20 monomers) complementary to the region of the gene where the mutation is desired is synthesized. One or more nucleotide substitutions are intentionally introduced into the central region of the oligonucleotide. The gene or fragment under investigation is cloned into an M13 phage-based vector to yield circular single-stranded recombinant DNA molecules. The recombinant vectors and oligonucleotides are then mixed and annealed. Hybridization of the oligonucleotide to its complementary region takes place, while non-complementary nucleotides remain unpaired. The oligonucleotide acts as a primer in an in vitro polymerization reaction mediated by DNA polymerase, and the circle is closed using ligases. The resulting circular molecule is introduced into E. coli cells, where partial repair of the mutant regions and replication take place. Mutation frequencies typically range from 1% to 50%. The selection of cells containing mutant DNA molecules can be accomplished through several methods, with preference given to techniques utilizing radioactively labeled oligonucleotides employed in the mutagenesis itself. In this scenario, the oligonucleotide serves as a probe. The principle behind such a probe relies on the fact that it is fully complementary to the mutant DNA and only partially complementary to the wild-type DNA. Hybridization conditions (primarily temperature) can be optimized so that the labeled probe forms a stable hybrid exclusively with the mutant DNA sequence, which can subsequently be detected via autoradiography.

Site-specific mutagenesis is particularly valuable because it enables the isolation of mutations without requiring monitoring of their phenotypic expression. This method opens up new avenues for investigating the functions of gene regulatory elements, adjusting promoter "strength", optimizing ribosome binding sites, and so forth. Protein Engineering represents one of the primary Applications of this methodology.

Protein engineering. This term refers to a set of methodological techniques that enable the reconstruction of a protein molecule through the targeted introduction of appropriate mutations into the structural gene (site-specific mutagenesis) and, consequently, the incorporation of desired Amino Acid Substitutions into the Primary Structure of the protein.

A striking example of engineering enhanced Proteins is provided by the experiments conducted by Fersht and colleagues on the enzyme tyrosyl-tRNA synthetase from the bacterium Bacillus stearothermophilus. Analyzing the consequences of amino acid substitutions within the Active Site of this enzyme led to the Conclusion that removing groups forming weak Hydrogen Bonds with the substrate can enhance its substrate affinity. Specifically, Threonine-51 (occupying position 51 in the peptide sequence) was found to form a long, weak Hydrogen bond with the ribose ring oxygen during the binding of tyrosyl adenylate. Conversely, Proline is located at the equivalent position in E. coli. Site-specific mutagenesis of the gene encoding B. stearothermophilus tyrosyl-tRNA synthetase enabled the thr-51→pro-51 substitution in the peptide. As a result, ATP binding within the enzyme's active site was dramatically improved, and its catalytic activity increased 25-fold.

Another equally significant example of protein redesign with practical implications is the modification of subtilisin from Bacillus amyloliquefaciens carried out by Estell and coworkers. Subtilisins are Serine proteinases secreted by bacilli into the extracellular environment. These enzymes are produced on a large scale by the biotechnology industry and are widely used as detergent components. A drawback of subtilisins is the drastic reduction in their proteolytic activity upon exposure to oxidizing agents, including those found in laundry detergents. The objective in redesigning the BPN' subtilisin molecule was to enhance its stability against chemical oxidation.

Preliminary experiments demonstrated that in the presence of hydrogen peroxide, subtilisin rapidly loses activity due to The oxidation of Methionine residue 222, which is converted to the corresponding sulfoxide. Site-specific mutagenesis was used to substitute this methionine residue with all 19 other standard Amino Acids. Plasmids harboring the mutant genes were introduced into strains with deletions in the corresponding genes, and The properties of the resulting subtilisins were analyzed. Mutants with serine and Alanine at position 222 proved to be sufficiently stable against hydrogen peroxide treatment. The mutant containing a Cysteine residue at position 222 exhibited the highest activity, with a specific activity 38% greater than that of the wild-type strain.

An analogous approach successfully enhanced The activity of ß-interferon. Other notable achievements in protein engineering include research elucidating the transforming activity of oncoproteins; altering enzyme thermostability, such as obtaining heat-labile renin and thermostable α-amylase; increasing the binding efficiency of Insulin to its Cell/33.html">Plasma Membrane receptor by replacing Histidine with aspartate at position 10 of the hormone's ß-chain; as well as numerous other Examples. A large number of protein-engineered products have already found Practical Applications in Industrial processes.