Simple Deletion: a vector- and marker-free method to generate and isolate site-directed deletion mutants

We have designed a new vector- and marker-free site-directed deletion system for gram-negative bacteria. In this system, a specific DNA fragment is amplified from a parental strain by using polymerase chain reaction (PCR), then circularized and introduced back into the parental strain for homologous recombination. The recombinant mutant is then detected and isolated by PCR-based sib selection. Unlike conventional methods, our Simple Deletion method requires no cloning procedures, and no foreign genes such as antibiotic-resistance genes are introduced as selection markers. The resulting mutant is, therefore, the same as the parental strain except for the lack of the target region. This method is categorized as a type of “self-cloning,” and the resulting mutant can be used for laboratory research without restrictions. Using this method, we generated a mutant of a plant pathogenic bacterium, Xanthomonas campestris pv. campestris, in which the 20.4-kb hrp gene cluster involved in the type III secretion system and in pathogenicity was deleted. In addition, we proved that this method can also be used to delete smaller DNA regions of X. campestris pv. campestris and to generate deletion mutants of the bacterium Ralstonia solanacearum.     

Role of the major capsid protein of phage T4 in DNA packaging from structure-function and site-directed mutagenesis studies

Heat cleavage of asp-pro peptide bonds was used to probe the primary structures of the Phage T4 major capsid protein precursor, gp23, its mature capsid form gp23*, and a DNA-dependent ATPase, called capsizyme. This analysis suggests that capsizyme is a gp23** resulting from the N-terminal processing found in gp23* as well as shortening at the C-terminus. Photoaffinity labeling with Azido-ATP and BrU-DNA, followed by heat cleavage, suggests binding sites for these compounds toward the C-terminus of gp23**, suggesting localization of functions within the gp23 primary sequence. Site-directed mutagenesis experiments were targeted therefore to the C-terminal end of g23 as well as to its processing sites. N-terminal processing site modification supports the consensus gp21 proteinase cleavage rule, whereas mutagenesis at the C-terminus suggests that the C-terminal alteration is unlikely to result from a gp21-morphogenesis proteinase cleavage. Amino acid replacements in gp23 at newly introduced amber sites reveal a new g23 mutant phenotype, defective partially DNA-filled heads, in support of the hypothesis that gp23 and its products function directly in the DNA packaging mechanism.     

An efficient method for multiple site-directed mutagenesis using type IIs restriction enzymes

Site-directed mutagenesis (SDM) methods are very important in modern molecular biology, biochemistry, and protein engineering. Here, we present a novel SDM method that can be used for multiple mutation generation using type IIs restriction enzymes. This approach is faster and more convenient than the overlap polymerase chain reaction (PCR) method due to its having fewer reaction steps and being cheaper than, but as convenient as, enzymatic assembly. We illustrate the usefulness of our method by introducing three mutations into the bacterial Streptococcus thermophilus Cas9 (bStCas9) gene, converting the humanized S. thermophilus Cas9 (hStCas9) gene into nuclease dead or H847A nickase mutants and generating sunnyTALEN mutagenesis from a wild-type TALEN backbone.     

Isolation of Cry1Ab protein mutants of Bacillus thuringiensis by a highly efficient PCR site-directed mutagenesis system

Abstract A site-directed mutagenesis method was designed and used to create Cry1Ab mutant proteins in two of the five highly conserved blocks present in the Cry protein family. Region 1 comprises the central α-helix 5 of domain I and has been implicated in the pore formation activity of the toxin. Substitution of arginine by serine at position 173 (R173S) affects neither structural integrity nor toxicity. Region 2 comprises the major part of the domain I/domain II interface, characterized by the presence of numerous hydrogen bonds and electrostatic interactions. Mutations in the salt bridge formed by aspartic acid 242 and arginine 265 (D242N, D242C, R265C, and D242C/R265C) resulted in structurally unstable mutant proteins as is shown by their increased protease sensitivity and lack of biological activity.     

A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR.

A rapid method is described to efficiently perform site-directed mutagenesis based on overlap extension polymerase chain reaction (OE-PCR). Two template DNA molecules in different orientations relative to only one universal primer were amplified in parallel. By choosing a high dilution of mutagenic primers it was possible to run an overlap extension PCR in only one reaction without purification of intermediate products. This method which we have named one-step overlap extension PCR (OOE-PCR) can in principle be applied to every DNA fragment which can be cloned into a multiple cloning site of any common cloning vector.     

TAMS technology for simple and efficient in vitro site-directed mutagenesis and mutant screening

Site-directed mutagenesis is an invaluable tool for functional studies and genetic engineering. However, most current protocols require the target DNA to be cloned into a plasmid vector before mutagenesis can be performed, and none of them are effective for multiple-site mutagenesis. We now describe a method that allows mutagenesis on any DNA template (eg. cDNA, genomic DNA and plasmid DNA), and is highly efficient for multiple-site mutagenesis (up to 100%). The technology takes advantage of the requirement that, in order for DNA polymerases to elongate, it is crucial that the 3′ sequences of the primers match the template perfectly. When two outer mutagenic oligos are incorporated together with the desired mutagenic oligos into the newly synthesised mutant strand, they serve as anchors for PCR primers which have 3′ sequences matching the mutated nucleotides, thus amplifying the mutant strand only. The same principle can also be used for mutant screening.     

An efficient system for site-directed mutagenesis to make various mutants of the env gene of human immunodeficiency virus type 1

We developed an efficient system of site-directed mutagenesis for the envelope (env) gene of human immunodeficiency virus type 1 (HIV-1). To make a template plasmid for mutagenesis, pS+B/MluI, two independent selection markers, i.e. a unique restriction site, MluI, and an in-frame termination codon, were introduced into the region encoding the V3 domain of the env gene of an HIV-1 strain, NL4-3, which had been cloned in the pUC118 plasmid. When the env gene of the pS+B/MluI plasmid was mutated successfully using mutagenic primers such as synthetic oligonucleotides or PCR-amplified DNA fragments longer than 1.5 kbp, the plasmids became resistant to digestion with MluI and competent env genes were formed by suppression of the in-frame termination. Various site-directed mutants of the env gene of HIV-1 were accurately constructed in a short time even in the absence of proper restriction sites by this system. The system of site-directed mutagenesis we reported here will be a useful method to analyze the functions of variable genes like the env gene of HIV-1 precisely and rapidly.     

Substitution of the conserved tryptophan 31 in Escherichia coli thioredoxin by site-directed mutagenesis and structure-function analysis

All prokaryotic and eukaryotic thioredoxins contain a conserved tryptophan residue, exposed at the active site disulfide/dithiol. The role of this W31 in Escherichia coli thioredoxin (Trx) was studied by site-directed mutagenesis. Four mutant Trx with W31Y, W31F, W31H, and W31A replacements were characterized. Very low tryptophan fluorescence emission from the remaining W28 was observed in all mutant Trx; reduction resulted in large, but variable increases (up to 11-fold) of fluorescence, to levels higher than in native or denatured wild-type Trx, demonstrating a previously postulated change involving W28. All W31 mutant Trx were good substrates for E. coli thioredoxin reductase. Compared with wild type, the apparent Km values were increased less than 2-fold for the W31A, W31H, and W31F Trx and the W31Y Trx showed even slightly higher catalytic efficiency (kcat/Km value). Functions of reduced Trx with ribonucleotide reductase and in reduction of insulin disulfides were more strongly influenced by the W31 replacements, in particular at low pH for A and H residues. T7 DNA polymerase activity generated by T7 gene 5 protein and reduced Trx was lowered by large factors for W31Y, W31A, or W31H compared with W31F or the wild-type protein. The in vivo function of Trx was studied by using pUC118-trxA expression in an E. coli trxA- background. The trxA genes with W31Y and W31F substitutions restored, fully and partly, the methionine sulfoxide utilization of a trxA- metE- test strain; W31A and W31H mutations resulted in no growth. Propagation of M13 was moderately impeded by W31Y and W31F or severely by W31A and W31H replacements. Growth of a phage T3/7 hybrid was possible only with the W31Y and W31F substitutions reflecting the in vitro results for T7 DNA polymerase.     

Production of thiol-penicillin-binding protein 3 of Escherichia coli using a two primer method of site-directed mutagenesis.

The active site serine residue of penicillin-binding protein 3 of Escherichia coli that is acylated by penicillin (Ser-307) has been converted to a cysteine residue using a simple and efficient two primer method of site-directed mutagenesis. The resulting thiol-penicillin-binding protein 3 was expressed under the control of the lacUV5 promoter in a high copy number plasmid. Constitutive expression of the thiol-enzyme (but not of the wild-type enzyme) was lethal, and the plasmid could only be maintained in E. coli strains that carried the lacIq mutation. Induction of the expression of the thiol-enzyme resulted in inhibition of cell division and the growth of the bacteria into very long filamentous cells. The inhibition of septation was probably due to interference of the function of the wild-type penicillin-binding protein 3 in cell division by the enzymatically inactive thiol-enzyme, and this implies that penicillin-binding protein 3 acts as part of a complex in vivo. We were unable to detect any acylation of the thiol-enzyme by penicillin, but it is not yet clear if this was because the thioester was not formed at an appreciable rate, or if it was formed but was too unstable to be detected by a modified penicillin-binding protein assay.     

Use of site-directed mutagenesis to obtain isomorphous heavy-atom derivatives for protein crystallography: cysteine-containing mutants of phage T4 lysozyme

Five different cysteine-containing mutants of the lysozyme from bacteriophage T4 were used to explore the feasibility of using site-directed mutagenesis to generate isomorphous heavy-atom derivatives for protein crystallography. Cysteines 54 and 97, present in wild-type lysozyme, can be readily reacted with mercuric ion to produce an excellent isomorphous heavy-atom derivative. Mutants with an additional cysteine at position 86, 146, 153 or 157, or with Cys 97 replaced by Val, were engineered by site-directed mutagenesis. The mutant lysozyme Thr 157----Cys reacts with mercuric chloride to give an excellent new derivative although Cys 157 is only approximately 60% substituted with the heavy atom. The cysteine at position 146 is largely buried but reacts readily with mercuric chloride. In this case the isomorphism is poor and the resultant derivative is of marginal quality. Cys 153 reacts rapidly with mercuric ion but the derivative crystals do not diffract. The mutant Pro 86----Cys does not yield a particularly good heavy-atom derivative. This is due in part to a loss of isomorphism associated with the mutation. In addition, Cys 86 shows very little reactivity towards mercurials even though it is fully exposed to solvent. The mutation Cys 97----Val was used to explore the possibility of creating an independent derivative by deleting a heavy-atom site already present in wild-type lysozyme. In all cases that were tested, the quality of the heavy-atom derivative was improved by using as an isomorphous pair mercury-substituted mutant versus non-substituted mutant rather than mercury-substituted mutant versus (non-substituted) wild-type lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simple and efficient oligonucleotide-directed mutagenesis using one primer and circular plasmid DNA template

A rapid and simple procedure for site-directed mutagenesis is described. This method uses only a single oligonucleotide primer with the double-stranded circular plasmid DNA as the template for mutagenesis. The phage T4 gene 32 product is included during primer extension in vitro to increase efficiency. Single and multiple changes as well as deletions have been obtained at an efficiency of 1–2%.     

A simple and efficient method for site-directed mutagenesis with double-stranded plasmid DNA.

A general, simple and efficient method for preparing site-specific mutations in double-stranded plasmid DNA without the need for special plasmids, bacterial strains or reagents is described. Only one synthetic oligonucleotide for each mutation is required, subcloning is unnecessary and a high efficiency of mutation (58-97%) was obtained. If two synthetic oligonucleotide primers are used, two separate mutations can be simultaneously created in a single reaction tube.     

Catalytic site targeted mutagenesis of the alpha-gingivain gene of Porphyromonas gingivalis using Tn-4351 to generate isogenic mutants

The extracellular proteinases of the anaerobe Porphyromonas gingivalis, are implicated in the destruction of host defence mechanisms in periodontitis. We have previously purified one of these enzymes, alpha-gingivain, and established that it belongs to the cysteine proteinase family of enzymes. In the present study, transposon Tn4351 was used to alter the open reading frame encoding a region that includes the catalytic site of alpha-gingivain by targeted mutagenesis. Escherichia coli HB101 which harbours R751 was used to introduce the transposon into P. gingivalis ATCC 33277 by conjugal transfer. E. coli was transformed using the altered plasmid with a Cla I site insertion of a sequence common to the catalytic site histidine or cysteine of many cysteine proteinases. The frequency of the transconjugation was 4.5 x 10(5) while the recipient viable counts comprised 60% of the original P. gingivalis. The result of this targeted mutagenesis was inactivation of gingivains such that some colonies on skimmed-milk agar plates showed no clear surrounding zones of hydrolysis and their normal catalytic activity towards L-BAPNA was destroyed.     

Further evidence that the CCK2 receptor is coupled to two transduction pathways using site-directed mutagenesis

A heterogeneity of CCK2 receptors has been reported which could correspond to different states of coupling to G proteins and/or association with different second messenger systems. To investigate these hypotheses, the wild-type CCK2 receptor and three mutants F347A, D100N and K333M/K334T/R335L, expected to modify the coupling of the G protein with the third intracellular loop of the receptor, were transfected into Cos-7 cells and their binding and signalling properties were evaluated using the natural ligand CCK8. Activation of wild-type as well as F347A, D100N or K333M/K334T/R335L CCK2 receptors by this ligand led to a similar arachidonic acid release which was blocked by pertussis toxin and the phospholipase A2 inhibitor, mepacrine. Nevertheless, in contrast to the wild-type CCK2 receptor, addition of CCK8 to cells transfected with the F347A or K333M/K334T/R335L mutants did not result in the production of inositol phosphates while the maximum increase in this second messenger formation was reduced by 30% with the D100N mutant. Taken together, these results suggest that the CCK2 receptor is coupled to two G proteins and that Phe347 and the cluster of basic residues K333/K334/R335 probably play a key role in Gq protein stimulation leading to inositol phosphate production but not in activation of the G protein coupled to phospholipase A2. These data bring additional support at the molecular level to the existence of different affinity states of CCK2 receptors suggested from the results of binding assays and behavioural studies.     

A QuikChange-like method to realize efficient blunt-ended DNA directional cloning and site-directed mutagenesis simultaneously

Here we present a QuikChange-like method to efficiently realize blunt-ended DNA cloning and conveniently introduce a site-directed mutation to recombinant plasmid at the same time. After blunt-ended DNA ligation and transformation, the plasmid DNA mixture is extracted from pooled transformants and directly used as template for PCR amplification with a pair of complementary mutagenic primers. With this method, sam1 gene was inserted into pUC19 vector by blunt-end ligation, and a unique restriction site Spe I was introduced to the recombinant plasmid at the same time. The randomly selected transformants were analyzed by DNA sequencing, and most of the clones were found to have correct sequences. However, no correct construct was found from randomly selected transformants after traditional blunt-ended DNA ligation and transformation.     

A site-directed mutagenesis method particularly useful for creating otherwise difficult-to-make mutants and alanine scanning

Site-directed mutagenesis has become routine in molecular biology. However, many mutants can still be very difficult to create. Complicated chimerical mutations, tandem repeats, inverted sequences, GC-rich regions, and/or heavy secondary structures can cause inefficient or incorrect binding of the mutagenic primer to the target sequence and affect the subsequent amplification. In theory, these problems can be avoided by introducing the mutations into the target sequence using mutagenic fragments and so removing the need for primer-template annealing. The cassette mutagenesis uses the mutagenic fragment in its protocol; however, in most cases it needs to perform two rounds of mutagenic primer-based mutagenesis to introduce suitable restriction enzyme sites into templates and is not suitable for routine mutagenesis. Here we describe a highly efficient method in which the template except the region to be mutated is amplified by polymerase chain reaction (PCR) and the type IIs restriction enzyme-digested PCR product is directly ligated with the mutagenic fragment. Our method requires no assistance of mutagenic primers. We have used this method to create various types of difficult-to-make mutants with mutagenic frequencies of nearly 100%. Our protocol has many advantages over the prevalent QuikChange method and is a valuable tool for studies on gene structure and function.     

Simple, quick and efficient site-directed antibody immobilization in a cartridge

A method is described for the simple, quick and efficient attachment of antibody within a cartridge for use as an immunoaffinity chromatography column. Antibodies are immobilized via their Fc regions through the use of periodate-oxidized carbohydrate functionalities of the immunoglobulin G. The method allows for the in situ coupling of the immunoglobulin G without prior removal of the oxidizing periodate solution. The entire procedure can be completed in 50 minutes. This method is especially useful for quick determinations of a particular monoclonal antibody's functionality or avidity towards a specific antigen. It may also be used in place of a conventional immunoaffinity column for the rapid isolation of small amounts of an antigen. This method will reduce the lengthy process of preparing an immunoaffinity column from several days to less than an hour.     

Dissection of the effector-binding site and complementation studies of Escherichia coli phosphofructokinase using site-directed mutagenesis

A systematic study by site-directed mutagenesis has been conducted on the effector site of phosphofructokinase from Escherichia coli to delineate the role of side chains in binding the allosteric activator, GDP, and inhibitor, PEP, and to search for key residues in the allosteric transtion. Target residues were identified from the crystal structure of the enzyme-nucleoside diphosphate complex. It is found that both activator and inhibitor bind to the same set of amino acid side chains. Deletion of positively charged groups (Arg21, Arg25, Arg54, Arg154, and Lys213 mutated to alanine) weakens binding of both effectors by 2-3 kcal/mol, consistent with the disruption of charged hydrogen bonds. Residue Glu187, which is known from the crystal structure to bind the coordinated Mg2+ ion of GDP, is found to have a unique behavior on mutation and appears to be crucial in triggering the allosteric transition. All other residues mutated simply weaken binding of both PEP and GDP in a parallel manner. However, mutation of Glu----Ala187 reverses the roles of GDP and PEP, causing GDP to become an allosteric inhibitor and PEP an activator. Mutation of Glu----Gln187 has only a small effect on the binding of PEP, and both PEP and GDP are inhibitors. Studies are described in which mutations in different subunits of a tetrameric complex complement each other. The effector site is composed of residues from two subunits. In particular, Arg21 and Lys213 in each site are from different subunits. Mutations of either one of these residues abolishes activation by GDP of the homotetramer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Turn scanning by site-directed mutagenesis: application to the protein folding problem using the intestinal fatty acid binding protein

We have systematically mutated residues located in turns between beta-strands of the intestinal fatty acid binding protein (IFABP), and a glycine in a half turn, to valine and have examined the stability, refolding rate constants and ligand dissociation constants for each mutant protein. IFABP is an almost all beta-sheet protein exhibiting a topology comprised of two five-stranded sheets surrounding a large cavity into which the fatty acid ligand binds. A glycine residue is located in seven of the eight turns between the antiparallel beta-strands and another in a half turn of a strand connecting the front and back sheets. Mutations in any of the three turns connecting the last four C-terminal strands slow the folding and decrease stability with the mutation between the last two strands slowing folding dramatically. These data suggest that interactions between the last four C-terminal strands are highly cooperative, perhaps triggered by an initial hydrophobic collapse. We suggest that this trigger is collapse of the highly hydrophobic cluster of amino acids in the D and E strands, a region previously shown to also affect the last stage of the folding process (Kim et al., 1997). Changing the glycine in the strand between the front and back sheets also results in a unstable, slow folding protein perhaps disrupting the D-E strand interactions. For most of the other turn mutations there was no apparent correlation between stability and refolding rate constants. In some turns, the interaction between strands, rather than the turn type, appears to be critical for folding while in others, turn formation itself appears to be a rate limiting step. Although there is no simple correlation between turn formation and folding kinetics, we propose that turn scanning by mutagenesis will be a useful tool for issues related to protein folding.