Efficient site-directed in vitro mutagenesis using phagemid vectors

Several methods have been developed that enhance the efficiency of in vitro, site-directed mutagenesis. Kunkel (8,9) has developed a method which uses a strong selection for the mutated strand and, hence, is highly efficient, but yet simple and rapid. This method originally used M13 phage as the vector. In this paper, we describe a refinement of this method using phagemid vectors, which combine the advantages of plasmids (such as high copy number and stability of cloned DNA) with the single-stranded DNA generating capability of M13 phage. We demonstrate that high efficiency of mutant production can be obtained with these vectors. We also analyzed by sequencing 11 mutated clones and found no second-site mutations, suggesting that alterations other than the site-directed mutation rarely occur in our system.     

Structure-function studies of human 5-alpha reductase type 2 using site directed mutagenesis

Site directed mutagenesis of human steroid 5alpha-reductase types 1 (5AR1) and 2 (5AR2) has been used to identify residues involved in inhibitor/substrate binding by 5AR2. Replacing residues 21-24 (GALA) in 5AR2 with the analogous residues 26-29 (AVFA) from 5AR1 did not significantly alter either the Km for testosterone or the Ki for the competitive inhibitor Finasteride. Replacement of AVFA in 5AR1 with GALA from 5AR2 however, significantly decreased the Km and increased the resistance to Finasteride. These findings confirm that 5AR1 residues 26-29 are involved in inhibitor/substrate binding but suggest residues 21-24 of 5AR2 are not. Replacing residues 20-29 (QCAVGCAVFA) of 5AR1 with the analogous residues 15-24 (ATLVALGALA) from 5AR2, changed the Km and Ki to values approaching those for wild type 5AR2. Replacing residues VAL in wild type 5AR2 with VGC from 5AR1 did not change Km or Ki but replacing ATL in 5AR2 with QCA from 5AR1 significantly decreased the Km and increased the resistance to Finasteride. Conversely, replacing QCA with ATL in 5AR1 containing GALA in place of AVFA, increased the Km and decreased resistance to Finasteride. These findings indicate residues 15-17 of human 5AR2 participate in inhibitor/substrate binding whereas residues 18-20 do not.     

Study of substrate specificity of human aromatase by site directed mutagenesis.

Human aromatase is responsible for estrogen biosynthesis and is implicated, in particular, in reproduction and estrogen-dependent tumor proliferation. The molecular structure model is largely derived from the X-ray structure of bacterial cytochromes sharing only 15-20% identities with hP-450arom. In the present study, site directed mutagenesis experiments were performed to examine the role of K119, C124, I125, K130, E302, F320, D309, H475, D476, S470, I471 and I474 of aromatase in catalysis and for substrate binding. The catalytic properties of mutants, transfected in 293 cells, were evaluated using androstenedione, testosterone or nor-testosterone as substrates. In addition, inhibition profiles for these mutants with indane or indolizinone derivatives were obtained. Our results, together with computer modeling, show that catalytic properties of mutants vary in accordance with the substrate used, suggesting possible differences in substrates positioning within the active site. In this respect, importance of residues H475, D476 and K130 was discussed. These results allow us to hypothesize that E302 could be involved in the aromatization mechanism with nor-androgens, whereas D309 remains involved in androgen aromatization. This study highlights the flexibility of the substrate-enzyme complex conformation, and thus sheds new light on residues that may be responsible for substrate specificity between species or aromatase isoforms.     

Efficient site-directed saturation mutagenesis using degenerate oligonucleotides.

We describe a reliable protocol for constructing single-site saturation mutagenesis libraries consisting of all 20 naturally occurring amino acids at a specific site within a protein. Such libraries are useful for structure-function studies and directed evolution. This protocol extends the utility of Stratagene's QuikChange Site-Directed Mutagenesis Kit, which is primarily recommended for single amino acid substitutions. Two complementary primers are synthesized, containing a degenerate mixture of the four bases at the three positions of the selected codon. These primers are added to starting plasmid template and thermal cycled to produce mutant DNA molecules, which are subsequently transformed into competent bacteria. The protocol does not require purification of mutagenic oligonucleotides or PCR products. This reduces both the cost and turnaround time in high-throughput directed evolution applications. We have utilized this protocol to generate over 200 site-saturation libraries in a DNA polymerase, with a success rate of greater than 95%.     

Genetic selection for a highly functional cysteine-less membrane protein using site saturation mutagenesis

We describe an efficient method for generating highly functional membrane proteins with variant amino acids at defined positions that couples a modified site saturation strategy with functional genetic selection. We applied this method to the production of a cysteine-less variant of the Crithidia fasciculata inosine-guanosine permease CfNT2 to facilitate biochemical studies using thiol-specific modifying reagents. Of 10 endogenous cysteine residues in CfNT2, two cannot be replaced with serine or alanine without loss of function. High-quality single- and double-mutant libraries were produced by combining a previously reported site saturation mutagenesis scheme based on the Stratagene Quikchange method with a novel gel purification step that effectively eliminated template DNA from the products. Following selection for functional complementation in Saccharomyces cerevisiae cells auxotrophic for purines, several highly functional noncysteine substitutions were efficiently identified at each desired position, allowing the construction of cysteine-less variants of CfNT2 that retained wild-type affinity for inosine. This combination of an improved site saturation mutagenesis technique and positive genetic selection provides a simple and efficient means to identify functional and perhaps unexpected amino acid variants at a desired position.     

Site-specific mutagenesis using synthetic oligodeoxyribonucleotide primers: II. In vitro selection of mutant DNA

A method for the in vitro selection of mutant DNA has been devised as an adjunct to the recently developed method for the use of short enzymatically-synthesized oligodeoxyribonucleotides of defined sequence as sitespecific mutagens for circular DNA. The selection method uses the mutating oligodeoxyribonucleotide as a primer for Escherichia coli DNA polymerase I (large fragment) under conditions where there is preferential interaction with mutant DNA template. After ligation using T4 DNA ligase, endonuclease Sl is used to degrade single-stranded non-mutant DNA leaving the desired mutant as closed circular duplex DNA. This paper describes the development of the method using mutants in ØX174 DNA as the model system. Studies on the changes A → G and G → A at position 587 of ØX174 viral DNA (am 3 to wild-type and its reversal) show that one or two cycles of selection can lead to a population of phage consisting of close to 100% mutants.     

Properties of a chimeric glucose dehydrogenase improved by site directed mutagenesis

Glucose dehydrogenase, a membrane bound enzyme oxidizing glucose to gluconic acid in the periplasmic space of Gram-negative bacteria plays a key role in mineral phosphate solubilization and is also an industrially important enzyme, being used as a glucose biosensor. A chimeric glucose dehydrogenase (ES chimera) encoding the N-terminal transmembrane domain from Escherichia coli and the C-terminal periplasmic domain from Serratia marcescens was constructed and the expression was studied on MacConkey glucose medium. The phosphate solubilizing ability of the chimeric GDH was also evaluated, substantiating the role of GDH in mineral phosphate solubilization (MPS). Four mutants of ES chimeric GDH were generated by site directed mutagenesis and the enzyme properties studied. Though the substrate affinity was unaltered for E742K and Y771M, the affinity of H775A and EYH/KMA to glucose and galactose decreased marginally and the affinity to maltose increased. Though Y771M showed a decreased GDH activity there was an increase in the heat tolerance. All the mutants showed an increase in the EDTA tolerance. The triple mutant EYH/KMA showed improved heat and EDTA tolerance and also an increase in affinity to maltose over the ES chimeric GDH.     

Site-directed mutagenesis using positive antibiotic selection

Various mutsgenesis protocols have been established that use the hybridization of a mismatched oligonucleotide to prime DNA synthesis on an M13 phagemid template. For efficient mutagenesis, all of these methods require a means to select for the mutant strand before or during amplification in anEscherichia coli host. In the Altered Sites II protocol, the mismatched oligonucleotide and an oligonucleotide that restores antibiotic resistance to the phagemid are simultaneously hybridized to the template and coupled by DNA synthesis and ligation. The restored antibiotic resistance is then used to select only those phagemids which incorporate the antibiotic repair oligonucleotide. Generally, between 60 and 90% of the phagemids recovered will incorporate both oligonucleotides. This method provides a simple an efficient technique for introducing specific mutations into DNA.     

Changes in binding activity of luteinizing hormone receptors by site directed mutagenesis of potential glycosylation sites.

Site directed mutagenesis of the rat ovarian luteinizing hormone (LH) receptor cDNA was performed at each of the six potential N-linked glycosylation sites to determine the effect of putative carbohydrate chains on the activity of the membrane receptor. The conversion of Asn173 to Gln resulted in the total loss of hormone binding to the surface of the transfected cell. Mutant receptors synthesized with substitutions at the remaining potential N-linked glycosylation positions of 77, 152, 269, 277 and 291 revealed no significant change in the hormone affinity. However Asn77Gln and Asn152Gln exhibited significant decreases (approximately 80%) in the number of high affinity hormone binding sites. The changes in hormone binding activity upon elimination of the potential glycosylation sites at 77, 152 and 173 indicate the presence of functional carbohydrate chains at these positions in the rat ovarian LH/hCG receptor.     

Solid phase in vitro mutagenesis using plasmid DNA template.

Site-specific mutagenesis was accomplished using a solid support to generate single stranded vector and insert fragments which can be used to form gap-duplex plasmids through flanking, complementary double stranded regions. More than 80% mutants were obtained in both a single and a double primer approach. No special vectors or strains are needed and mismatch repair is avoided as the mutagenesis region is in a single stranded form when transformed into the Escherichia coli host cell. The fragments to be immobilized can be produced either by a polymerase chain reaction using general primers or by a site-specific restriction followed by a fill-in reaction. This novel method is rapid, simple and flexible and well suited for both manual and semi-automated in vitro mutagenesis protocols.     

Large-scale mutagenesis directed at specific chromosomes in wheat.

A novel approach has been developed to allow for the efficient selection of loss-of-function wheat mutants in the M1 generation, following either physical or chemical mutagenesis. This has generated an order of magnitude increase in the efficiency of identification of mutants, and also greatly increases the likelihood that selected individuals reflect mutation events at the target locus, rather than at genes acting elsewhere in the disease resistance pathway. The approach relies only on prior knowledge of the chromosomal location of the target gene, and uses the polyploidy of wheat to construct populations for mutagenesis in which large numbers of individuals are hemizygous for the target gene. The idea is illustrated with the mass identification of mutants at three independent genes for race-specific resistance to yellow rust, and one gene for resistance to powdery mildew.     

insilico.mutagenesis: a primer selection tool designed for sequence scanning applications used in directed evolution experiments

Several primer prediction programs have been developed for a variety of applications. However none of these tools allows the prediction of a large set of primers for whole gene site-directed mutagenesis experiments using the megaprimer method. We report a novel primer prediction tool (insilico.mutagenesis), accessible at www.insilico.uni-duesseldorf.de, developed for the application to high-throughput mutagenesis used in directed evolution or structure-function dependency projects, which involve the subsequent mutagenesis of a large number of amino acid positions (e.g., in whole gene saturation or gene scanning mutagenesis experiments). Furthermore, the program is suitable for all site-directed (saturation) mutagenesis approaches, such as saturation mutagenesis of promoter sequences and other types of untranslated intergenic regions. In anticipation of downstream cloning steps, the primer design tool also includes a restriction site control feature alerting the user if unwanted restriction sites have been introduced within the mutagenesis primer. The use of our tool promises to speed up the process of site-directed mutagenesis, as it instantly allows predicting a large set of primers.     

Biosynthesis of hepatitis B virus e antigen: directed mutagenesis of the putative aspartyl protease site.

The C gene products of all mammalian hepadnaviruses contain a region with sequence similarities to the catalytic center of the aspartyl proteases. This region could have the capacity to cleave precore proteins, leading to the synthesis of e antigen. By site-directed mutagenesis on a plasmid containing the hepatitis B virus C gene, we have replaced either the Asp residue of the putative aspartyl protease catalytic center or an Asp residue located 3 amino acids upstream. Transient expression of the mutated hepatitis B virus C gene in human and mouse cells showed that none of these mutations prevented the secretion of an accurately processed HBe antigen. Thus, we demonstrated that the aspartyl protease responsible for e antigen precursor processing is not C gene encoded but is more likely to be a cellular enzyme. From these results, we suggest a model for the mechanism of e antigen synthesis.     

Structure-function analysis of epidermal growth factor: site directed mutagenesis and nuclear magnetic resonance

The role of leucine-47 in determining the structure and activity of human epidermal growth factor was examined using site-directed mutagenesis. Wild type protein and four variants in which Leu47 was replaced by valine, glutamate, aspartate and alanine were produced from yeast. 1H NMR experiments demonstrated that substitution of Leu47 had little effect on the protein structure. The observed reduction in receptor binding affinity caused by the substitutions could thus be attributed to perturbation of a residue directly involved in receptor interactions.     

Investigation of the substrate recognition of Drosophila melanogaster nucleoside kinase by site directed mutagenesis

The deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) has a broad substrate specificity and a higher catalytic rate than other known deoxyribonucleoside kinases. Therefore it is a natural candidate for possible use as a suicide gene in combined gene/chemotherapy of cancer. We have performed site directed mutagenesis and tested different truncated forms of the enzyme in order to increase the affinity for ganciclovir.     

Retroviral integration: in vitro host site selection by avian integrase.

Viral integrase catalyzes the integration of the linear viral DNA genome into the chromatin of the infected host cell, an essential step in the life cycle of retroviruses. The reaction produces a characteristic small duplication of host sequences at the site of integration, implying that there is a close juxtaposition of the viral DNA ends during a concerted integration event. We have used an in vitro assay to measure the concerted integration of virus-like plasmid DNA into naked lambda DNA catalyzed by virion purified avian integrase. In contrast to in vivo avian integration, which has strong fidelity for a 6-bp duplication, purified avian integrase in the context of this assay produced a distribution of duplication sizes, with the 6-bp size dominating. The metal cofactor Mg2+ induced increased fidelity for the 6-bp duplication relative to that with Mn2+. The immediate sequence of the host site may also influence duplication size in that we found sites that sustained multiple independent integration events producing the same duplication size. Additionally, for each set of cloned integration sites (5, 6, and 7 bp), a unique but similar symmetrical pattern of G/C and A/T sequence biases was found. Using duplex oligonucleotides as target substrates, we tested the significance of the 6-bp G/C and A/T pattern for site selection. In the context of this assay, which is likely dominated by the integration of only one viral end, the 6-bp pattern was not preferred. Instead, integration was predominantly into the 3' ends of the oligonucleotides. The combined results of the lambda and oligonucleotide assays indicated that although host site selection has properties in common with recognition of the viral DNA termini, the nonrandom sequence preferences seen for host site selection were not identical to the sequence requirements for long terminal repeat recognition.     

Studies of the melatonin binding site location onto quinone reductase 2 by directed mutagenesis

Melatonin is a neurohormone implicated in both biorhythm synchronization and neuroprotection from oxidative stress. Its functions are mediated by two G-protein-coupled-receptors (MT1 and MT2) and MT3, which corresponds to quinone oxidoreductase 2 (QR2). To determine the binding site of QR2 for melatonin, point mutations of residues crucial for the enzymatic activity of hQR2 were performed. The substitution of the hydrophobic residues Phe126, Ile128 and Phe178 by tyrosines at the active site significantly increased enzymatic activity and decreased the affinity of a structural analog of melatonin, the 2[¹²⁵I]iodo-MCANAT. The mutation of residues implicated in zinc chelating (His¹⁷³; His¹⁷⁷) had no effect on radioligand binding. Destabilisation of the cofactor FAD by mutation N18E showed that 2[¹²⁵I]iodo-MCANAT binding was closely linked to the conformational integrity of human QR2. Surprisingly, the mutations C222F and N161A, which are distant from the determined binding site of the ligand, increased the affinity of 2[¹²⁵I]iodo-MCANAT for hQR2. What seems to better explain the binding variations among the mutants are the activity recorded with BNAH and coenzyme Q1. Various hypotheses are discussed based on the various parameters used in the study: nature of the substrates and co-substrates and nature of the amino acid changes. This study, which constitutes the first structural analysis of hQR2, should enable to better understand the biological role of melatonin on this enzyme and particularly, the discrepancies between the pharmacologies of the melatonin binding site (MT3) and the QR2 catalytic activity.