Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Fundamentals of Molecular Biotechnology
Site-Directed Mutagenesis and Protein Engineering
Site-Directed Mutagenesis: Methodology

Recombinant DNA technology makes it possible to isolate genes for any naturally occurring Proteins, express them in a specific host Organism, and obtain pure protein products. However, the physical and Chemical properties of such “natural” proteins often fail to meet the requirements for industrial Applications. Sometimes, to obtain proteins with desired properties, researchers use organisms that grow under unusual, often extreme conditions as a source of the corresponding genes. For example, to synthesize an α-amylase that retains activity at high temperatures, its Gene was isolated from Bacillus stearothermophilus, a bacterium naturally inhabiting hot springs with a Water Temperature of 90 °C. The α-amylase obtained in this way remained active at the temperatures used in the industrial production of ethanol from starch. Mutant forms of genes can also be used to produce proteins with predefined properties. However, the number of mutant proteins resulting from single nucleotide substitutions in a structural gene via conventional mutagenesis is extraordinarily large. Mutagenesis followed by screening rarely leads to a significant improvement in The properties of the parent protein, because most Amino Acid Substitutions are accompanied by a decrease in enzyme activity.

To design proteins with specific properties, another approach can be used, based on introducing targeted modifications into their cloned coding genes. This makes it possible to obtain proteins with properties distinct from those of their natural analogs.

✵ By modifying the Michaelis constant (K_M), which characterizes the binding affinity of the substrate for the enzyme, and the maximum velocity (V_max) of substrate conversion into product under specific conditions, the overall catalytic efficiency of the reaction can be enhanced; V_max is equal to the total Enzyme Concentration (E₀) multiplied by the catalytic constant (k_cat).

✵ By increasing protein stability over a wide range of temperatures or pH levels, it can be utilized under conditions where the wild-type protein would be inactivated.

✵ By engineering proteins capable of functioning in anhydrous Solvents, catalytic reactions can be carried out under non-physiological conditions.

✵ By modifying a protein to function independently of a cofactor, it can be successfully employed in various continuous Industrial processes.

✵ Modifying the Active Site of an enzyme can enhance its Specificity and reduce unwanted Side Reactions.

✵ Increasing the resistance of a protein to cellular proteases can simplify its purification Procedure and increase product yield.

✵ Altering the Allosteric Regulation of an enzyme can reduce the degree of its feedback inhibition by a metabolite and increase product yield.

Designing a novel protein with predetermined properties is a challenging task, yet modifying an existing protein is entirely feasible. Changes can be introduced directly into the protein or into its gene. However, Chemical modification of proteins is rarely strictly specific and must be repeated for each individual protein preparation; therefore, it is preferable to modify its cloned gene. Unfortunately, it is not always clear which specific amino acid or Amino Acid Sequence needs to be altered to achieve the desired physical, kinetic, or chemical properties. It may turn out that modifications must affect two or more amino acid residues that are far apart in the polypeptide chain but are brought into close proximity As a result of protein folding. It is hoped that in the near future, computer modeling will enable the prediction of a protein's properties based solely on its amino acid sequence. This will greatly facilitate the design of tailor-made proteins. Introducing novel Genetic information into cloned genes is relatively straightforward nowadays; however, to determine whether the target protein possesses the desired properties, numerous protein variants must be screened.

The Introduction of specific changes into DNA coding sequences that result in targeted alterations of Amino acid sequences is known as site-directed mutagenesis. Identifying which Amino Acids to substitute to achieve the desired outcome is facilitated when the three-dimensional Structure OF THE protein is known in detail (determined via X-ray crystallography or other Analytical Methods). However, such data are unavailable for the majority of proteins; consequently, site-directed mutagenesis remains largely an empirical, trial-and-error procedure. Each protein encoded by a mutant gene must be tested to confirm that the mutation has produced the desired effect.

Various experimental approaches are employed for the site-directed mutagenesis of cloned genes. In some cases, specific sites within the cloned gene are targeted for modification; in others, a short fragment of the cloned gene is mutated randomly, and mutant proteins possessing the requisite activity are subsequently screened and selected.

Oligonucleotide-directed mutagenesis using M13 phage DNA

Oligonucleotide-directed (site-specific) mutagenesis is one of the simplest methods for introducing point Mutations into a cloned gene (Fig. 8.1). To carry out this procedure, one must know: (1) the exact nucleotide sequence of the DNA region that corresponds to the mRNA codon to be altered; and (2) The Nature of The amino acid replacements. Typically, the target gene is inserted into a double-stranded form of an M13-based bacteriophage vector. First, the single-stranded form of the vector (the M13 plus strand) is isolated and mixed with a synthetic oligonucleotide that is perfectly complementary—except for a single nucleotide—to the desired segment of the cloned gene. This differing (i.e., mismatched) nucleotide corresponds to the nucleotide of the mRNA codon that needs to be changed. In the example shown in Fig. 8.1, the triplet ATT, which specifies the isoleucine codon AUU, is to be replaced by the triplet CTT, corresponding to the leucine codon CUU. The oligonucleotide will hybridize with the complementary region of the cloned gene provided that: (1) it is added in vast excess relative to the M13 DNA; (2) the mismatched nucleotide is located approximately in the middle of the oligonucleotide; and (3) annealing is performed at a low temperature and high Ionic strength. The 3'-end of the hybridized oligonucleotide serves as a primer to initiate DNA Synthesis, while the intact M13 DNA strand acts as a template. Replication is catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I in the presence of all four deoxyribonucleoside triphosphates, and the joining of the last nucleotide of the synthesized strand to the 5'-end of the primer is mediated by T4 phage DNA ligase. However, in vitro DNA synthesis rarely goes to completion, and the partially double-stranded molecules must be separated from normal ones by sucrose gradient centrifugation.

E. coli Cells are then transformed with fully double-stranded M13 phage DNA molecules that nevertheless contain non-complementary NUCLEOTIDES. Inside the cells, phage particles are produced, ultimately leading to Cell lysis and plaque formation. Because replication proceeds via a semi-conservative mechanism, half of the resulting phage particle population should contain wild-type DNA, while the other half contains mutant DNA with the specific nucleotide substitution. Particles containing exclusively the mutant gene are identified by stringent DNA Hybridization using the original oligonucleotide as a probe. The mutant gene is excised and inserted into an appropriate E. coli expression vector. Finally, the mutant protein is synthesized in E. coli and purified.

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Fig. 8.1. Oligonucleotide-directed mutagenesis. Single-stranded M13 phage DNA (plus strand) carrying the target gene is annealed with a complementary synthetic oligonucleotide that contains a single base mismatch compared to the original DNA template. The oligonucleotide acts as a primer for DNA synthesis, and the M13 vector with the inserted gene serves as the template. Replication is catalyzed by the E. coli DNA polymerase I Klenow fragment. The synthesized full-length strand is circularized by T4 DNA ligase. E. coli is transformed with the resulting double-stranded molecules. Some of the phage progeny contain wild-type DNA, whereas others contain mutant DNA.

In practice, the proportion of phage particles carrying the mutant gene is much lower than the expected 50%: only 1–5% of the plaques contain the phage with the mutant gene. To improve the recovery of mutant phage, the oligonucleotide-directed mutagenesis procedure was modified. One approach involved introducing the M13 vector carrying the gene to be mutated into an E. coli strain deficient in two Enzymes of DNA METABOLISM (Fig. 8.2). The first enzyme is a mutant form of dUTP pyrophosphatase (dut). Cells with inactive dUTP pyrophosphatase exhibit elevated intracellular levels of dUTP, which leads to the incorporation of several dUTP residues in place of dTTP during replication. The second enzyme is defective uracil-N-glycosylase (ung). In the absence of functional uracil-N-glycosylase, dUTP residues randomly incorporated into DNA cannot be excised. In single-stranded M13 DNA synthesized in such E. coli cells, approximately 1% of the thymidine residues are replaced by uridine. The mismatched oligonucleotide is annealed with the uracil-containing M13 DNA, and the second strand is synthesized in vitro. The double-stranded DNA is then used to transform an E. coli strain harboring a functional ung gene. Active host uracil-N-glycosylase removes the uridine residues from the M13 DNA (Fig. 8.2), resulting in the degradation of the original template strand, leaving only the dUTP-free mutant strand to be replicated. As a result, the yield of phage particles carrying the mutant gene is significantly increased.

Oligonucleotide-directed mutagenesis using plasmid DNA The main drawback of oligonucleotide-directed mutagenesis using M13 phage is the large number of procedural steps involved. Isolating the mutant form of the desired gene is time-consuming. As an alternative to the M13 system, numerous other approaches based on plasmid DNAs have been developed. This eliminates the need to transfer the gene of interest from a plasmid into phage DNA and, following mutagenesis, back into a plasmid. One such approach involves inserting the DNA into a plasmid vector that carries a functional tetracycline resistance gene and an inactive ampicillin resistance gene containing a single-nucleotide substitution in the middle of the gene (Fig. 8.3). E. coli cells are transformed with the vector carrying the target DNA, and the double-stranded plasmid DNA is denatured with alkali to yield single-stranded circular molecules. The denatured DNA is annealed with three different oligonucleotides. One of them is designed to introduce the desired change into the cloned target gene, the second to repair the mutation in the ampicillin resistance gene, and the third to introduce a single-nucleotide substitution that inactivates the tetracycline resistance gene. Four deoxyribonucleoside triphosphates and T4 DNA polymerase—which Functions analogously to the E. coli DNA polymerase I Klenow fragment—are added to the reaction mixture. The hybridized oligonucleotides serve as primers for DNA synthesis, using the intact circular DNA molecule as a template. Nicks in the newly synthesized strand are sealed with T4 DNA ligase. Upon completion of synthesis and ligation, the reaction products are used to transform E. coli cells. Transformants are selected for ampicillin resistance and tetracycline sensitivity. Approximately 90% of these transformants carry the specific mutation in the cloned gene. In the remaining transformants, the cloned gene remains unaltered, either because the oligonucleotide failed to hybridize or because it was displaced during DNA synthesis. Cells carrying the mutated cloned gene are identified by hybridization. All Plasmids, strains, enzymes, oligonucleotides (except the mutagenic one specific for the cloned gene), and buffers are commercially available as kits to simplify the procedure.

Fig. 8.2. Increasing the yield of mutant M13 phage by transformation of an E. coli dut ung strain. The target gene is cloned into the double-stranded replicative form of M13 phage DNA, and the resulting molecules are used to transform an E. coli dut ung strain. The dut mutation causes an elevated intracellular level of dUTP, leading to the incorporation of multiple dUTP residues (U) into the DNA, while the ung mutation blocks their removal. Wild-type E. coli cells are transformed with the double-stranded M13 DNA containing the target gene. Wild-type ung gene product (uracil-N-glycosylase) removes all uracil residues from the original strand, causing its degradation. The mutant strand remains intact because it lacks uracil residues. This strand serves as a template for DNA replication, thereby increasing the proportion of phage particles carrying the mutant gene.

Fig. 8.3. Oligonucleotide-directed mutagenesis using plasmid DNA. The target gene is inserted into the polylinker of the pALTER vector. Plasmid DNA is alkali-denatured and annealed with three oligonucleotides: a mutagenic oligonucleotide, an oligonucleotide that restores ampicillin resistance (Ampr), and an oligonucleotide that confers tetracycline sensitivity (Tet^s). These oligonucleotides act as primers for DNA synthesis catalyzed by T4 DNA polymerase, with the original strand serving as the template. Single-stranded nicks in the newly synthesized strand are sealed by T4 DNA ligase. E. coli cells are transformed with the reaction products, and Amp^r Tet^s transformants are selected.

Oligonucleotide-directed mutagenesis using PCR Amplification

A simpler and faster alternative to the M13 system for producing large quantities of mutant genes is site-specific mutagenesis combined with the Polymerase Chain Reaction (PCR). One variation of this approach is as follows. The target gene is inserted into a plasmid vector (Fig. 8.4), and the sample is divided into two tubes. Two specific PCR primers are added to each tube: primers 1 and 2 to one tube, and primers 3 and 4 to the other. Primers 2 and 3 are fully complementary to a region of the cloned gene or its flanking sequence, whereas primers 1 and 3 are complementary to another region but contain a single mismatched nucleotide and hybridize to opposite strands, thereby introducing a substitution in both nucleotides of the given base pair. The binding sites of primers 1 and 2 in one tube and primers 3 and 4 in the other are positioned such that the PCR products in the two tubes have different ends. After the PCR amplification, the contents of both tubes are mixed, denatured, and allowed to reanneal. Because the ends of the amplified DNA molecules from the two tubes differ, single-stranded DNAs from the different tubes associate to form circular molecules containing two single-stranded nicks. These nicks are repaired in vivo following E. coli transformation. Reannealing of single strands originating from the same tube generates linear molecules. Only circular molecules, rather than linear ones, are stably maintained as plasmids and inherited in E. coli cells, and all of them carry the site-specific mutation. Thus, this method allows point mutations to be introduced into a cloned gene without the need to insert the gene into M13 phage DNA, use dut ung mutant E. coli strains, or transfer the mutant gene from an M13 vector into an expression vector.

Random Mutagenesis Using Degenerate Oligonucleotide Primers

Unfortunately, it is usually unknown beforehand which nucleotide substitution in a cloned gene is required to yield a protein with desired properties. Therefore, researchers often have to alter a specific nucleotide site in every possible way. For instance, one can synthesize oligonucleotide primers that contain different nucleotides at one of the target sites. Such "degenerate" oligonucleotides are typically produced by adding a small amount (up to several percent) of the other three nucleotides into the automated DNA synthesizer at a specific stage when a designated nucleotide is supposed to be incorporated into the chain (Fig. 8.5). As a result, this yields a heterogeneous set of oligonucleotide primers for a single site, which can be used to generate a corresponding pool of mutant target genes featuring nucleotide substitutions at the specific site.

This approach offers two major advantages: 1) there is no need to precisely know the specific role that a particular amino acid residue plays in protein function; 2) because various amino acid substitutions occur at this site, proteins with a wide range of interesting and useful properties may be synthesized serendipitously. Naturally, if none of the resulting proteins exhibit the desired characteristics, the entire process must be repeated by synthesizing a new set of degenerate primers complementary to a different region of the gene.

Fig. 8.4. Oligonucleotide-directed mutagenesis using PCR. The reaction is carried out in two separate tubes, each containing identical double-stranded plasmid DNA but different sets of primers. Primers 1 and 3 contain a single mismatched nucleotide and are complementary to different strands of the plasmid DNA. Primers 2 and 4 are fully complementary to the corresponding Regions of the plasmid DNA and also hybridize with opposite strands. The hybridization sites for each primer pair differ in position, but their ends abut. PCR amplification yields linear molecules. Upon completion of the reaction, the Contents of the tubes are mixed, denatured, and then allowed to reanneal. Consequently, alongside the two original amplified linear molecules, two circular plasmid DNA molecules are formed, each containing two single-stranded nicks. Following Transformation of E. coli cells with these circular molecules, the nicks are repaired by host cell enzymes, allowing the plasmid to replicate independently. Linear DNA molecules are not maintained in E. coli.

Partially degenerate oligonucleotides can be integrated into a target gene in several ways. One approach is as follows. The gene is inserted into a plasmid between two unique restriction sites, and its overlapping left and right fragments are amplified using multiple PCR reactions (Fig. 8.6). The primer pair used to amplify the left fragment includes an incompletely complementary oligonucleotide that pairs with the heavy strand of the target gene, and a standard, fully complementary primer that hybridizes with the light-strand region flanking the left unique restriction site. One of the primers used for the amplification of the right fragment contains non-complementary nucleotides and pairs with the heavy strand of the target gene, whereas the second primer is fully complementary to the light-strand region flanking the second (right) unique restriction site. The PCR amplification products are purified, combined, and then subjected to Denaturation and reannealing. This generates a fraction of partially double-stranded DNA molecules paired across the target gene region. These are extended into fully double-stranded molecules using DNA polymerase, followed by PCR amplification with a pair of primers complementary to the opposite ends of the molecules. The amplified products are digested with two Restriction Endonucleases whose unique sites are located at the ends of the fragment, and then ligated into an appropriate plasmid vector. This approach enables the generation of modified genes carrying random mutations.

Fig. 8.5. Chemical Synthesis of Oligonucleotide primers containing different nucleotides at designated sites. In this case, the vessel containing G-phosphoramidite (94%) is also supplemented with A (2%), C (2%), and T (2%) phosphoramidites, so that the reaction yields a mixture of oligonucleotides where A, C, or T are present at the positions designated for G.

Fig. 8.6. Random mutagenesis using degenerate oligonucleotides and PCR. The left and right portions of the target gene are amplified separately via PCR. The respective primers are indicated by horizontal arrows. Degenerate oligonucleotides are depicted as arrows with three barbs, each corresponding to a nucleotide that is non-complementary to the matching nucleotide in the target gene. The amplified fragments are purified, denatured to achieve complete strand Separation, and reannealed. This produces partially double-stranded DNA molecules paired in the region of the target gene. They are extended using DNA polymerase and subjected to PCR amplification. The PCR products are digested with restriction endonucleases A and B and inserted into a vector treated with the same enzymes.

Random Mutagenesis Using Nucleotide Analogues

In addition to Methods of Introducing mutations into a cloned gene based on M13 phage, other approaches utilizing plasmid DNA have been developed. One of these is schematically illustrated in Fig. 8.7. The target gene is inserted into a plasmid near two closely spaced restriction sites. These sites are selected such that Digestion with the two restriction enzymes produces recessed 3'- and 5'-ends—specifically, the 3'-end of the restriction site adjacent to the cloned gene is recessed, while the 3'-end on the opposite side of the plasmid protrudes. E. coli exonuclease III (ExoIII) digests DNA exclusively from recessed 3'-ends, but not from protruding 3'-ends or any 5'-ends. It is added to the reaction mixture following incubation of the DNA with the two restriction enzymes, and it progressively cleaves nucleotides one by one from the recessed 3'-end of the strand. After a predetermined time, the reaction is stopped, and the gap is filled using the Klenow fragment of DNA polymerase I in the presence of a mixture of the four standard deoxynucleotides supplemented with an analogue of one of them. This yields plasmids containing the target gene with the analogue incorporated at one or more sites. These plasmids are then used to transform E. coli cells. Upon replication, a nucleotide different from that in the original gene is incorporated into the cloned gene. Besides the method described above, other techniques are also employed for random mutagenesis, such as a variant of oligonucleotide-directed mutagenesis using M13 phage DNA. In this case, DNA synthesis is primed using a mixture of oligonucleotides carrying random substitutions. This generates clone libraries harboring numerous mutations at various sites. A major limitation of approaches that introduce a high frequency of random mutations into a cloned gene is the necessity of screening every clone to identify the one directing the Synthesis of the desired protein. Although this is a challenging task, it is frequently the only way to discover proteins with novel properties. Once this goal is achieved, The nucleotide sequence of the corresponding cloned gene is determined, and the mutated site(s) identified.

Fig. 8.7. Introduction of random mutations into a cloned gene. The vector carrying the cloned gene is digested with restriction enzymes RE1 and RE2, generating one recessed 3'- and one recessed 5'-end (and correspondingly, one protruding 3'- and one protruding 5'-end). It is then treated with ExoIII, which digests DNA exclusively from the recessed 3'-end, removing nucleotides one at a time. After a brief incubation, the reaction is terminated, and the resulting gap is filled using the Klenow fragment of E. coli DNA polymerase I. All four deoxynucleoside triphosphates (dNTPs) along with a small amount of an analogue of one of them are added to the reaction mixture. The product is treated with nuclease S1 to generate blunt ends, ligated using T4 DNA ligase, and used to transform E. coli cells. During subsequent vector DNA replication, nucleotides differing from the original ones are incorporated into the complementary strand at the position where the nucleotide analogue resides, thereby introducing a mutation into the cloned gene.