Site-directed Mutagenesis Method Research Articles

Improving the Thermostability and pH Stability of Aspergillus niger Xylanase by Site-directed Mutagenesis

To increase the thermostability and pH stability of the recombinant xylanase MxynB from the Aspergillus niger nl-1, site-directed mutagenesis method was selected by rational approaches based on the three-dimensional structures using a homology modeling of mxynB constructed by SWISS-MODEL and BLAST. The effect of disulphide in the structure of xylanase was studied to increase the optimum temperature and thermostability of enzyme, the variant MxynB-116-135 which was created with amino acid residues substitutions G116C+Y135C by site-directed mutagenesis had its reaction temperature for the maximum activity at 50oC, which corresponded to a basic 10oC unit increase compared with the wild-type enzyme. Moreover, the thermostability of A. niger nl-1 xylanase after mutation MxynB-G116C-Y135C-D76R-N28H-N29D-Y45M-N47L was clearly increased by 570% as compared with the wild-type enzyme under 70oC. In addition, the optimal activity pH of the enzyme after the mutations MxynB-G116C-Y135C-D76R-N28H-N29D-Y45M-N47L and MxynB-G116C-Y135C-S58H-D76R-N28H-N29D-Y45M-N47L raised from 4.5 to 6.0, meanwhile, the pH stabilities were increased from 120 to 194% compared with the wild-type enzyme under pH 5.0 to 7.0. All the results suggest that these amino acid residues are important in determining the thermostability and pH stability of MxynB from Aspergillus niger nl-1, which will raise its potential interest for the industrial applications.

A signalling cascade involving receptor-activated phospholipase A2, glycerophosphoinositol 4-phosphate, Shp1 and Src in the activation of cell motility

BackgroundShp1, a tyrosine-phosphatase-1 containing the Src-homology 2 (SH2) domain, is involved in inflammatory and immune reactions, where it regulates diverse signalling pathways, usually by limiting cell responses through dephosphorylation of target molecules. Moreover, Shp1 regulates actin dynamics. One Shp1 target is Src, which controls many cellular functions including actin dynamics. Src has been previously shown to be activated by a signalling cascade initiated by the cytosolic-phospholipase A2 (cPLA2) metabolite glycerophosphoinositol 4-phosphate (GroPIns4P), which enhances actin polymerisation and motility. While the signalling cascade downstream Src has been fully defined, the mechanism by which GroPIns4P activates Src remains unknown.MethodsAffinity chromatography, mass spectrometry and co-immunoprecipitation studies were employed to identify the GroPIns4P-interactors; among these Shp1 was selected for further analysis. The specific Shp1 residues interacting with GroPIns4P were revealed by NMR and validated by site-directed mutagenesis and biophysical methods such as circular dichroism, isothermal calorimetry, fluorescence spectroscopy, surface plasmon resonance and computational modelling. Morphological and motility assays were performed in NIH3T3 fibroblasts.ResultsWe find that Shp1 is the direct cellular target of GroPIns4P. GroPIns4P directly binds to the Shp1-SH2 domain region (with the crucial residues being Ser 118, Arg 138 and Ser 140) and thereby promotes the association between Shp1 and Src, and the dephosphorylation of the Src-inhibitory phosphotyrosine in position 530, resulting in Src activation. As a consequence, fibroblast cells exposed to GroPIns4P show significantly enhanced wound healing capability, indicating that GroPIns4P has a stimulatory role to activate fibroblast migration. GroPIns4P is produced by cPLA2 upon stimulation by diverse receptors, including the EGF receptor. Indeed, endogenously-produced GroPIns4P was shown to mediate the EGF-induced cell motility.ConclusionsThis study identifies a so-far undescribed mechanism of Shp1/Src modulation that promotes cell motility and that is dependent on the cPLA2 metabolite GroPIns4P. We show that GroPIns4P is required for EGF-induced fibroblast migration and that it is part of a cPLA2/GroPIns4P/Shp1/Src cascade that might have broad implications for studies of immune-inflammatory response and cancer.

High-Throughput Site-Directed Mutagenesis.

Protein engineering has an array of uses: whether you are studying a disease mutation, removing undesirable sequences, adding stabilizing mutations for structural purposes, or simply dissecting protein function. Protein engineering is almost exclusively performed using site-directed mutagenesis (SDM) as this provides targeted modification of specific amino acids, as well as the option of rewriting the native sequence to include or exclude certain regions. Despite its widespread use, SDM has often proved to be a bottleneck, requiring precision manipulation on a sample-by-sample basis to make it work. When dealing with large volumes of samples it is not possible to use such a low-throughput approach. Here we describe a high-throughput (HTP) method for SDM, optimized and used by the Structural Genomics Consortium (SGC) to complement structural studies.

Site-selective protein conjugation at histidine.

Site-selective conjugation generally requires both (i) molecular engineering of the protein of interest to introduce a conjugation site at a defined location and (ii) a site-specific conjugation technology. Three N-terminal interferon α2-a (IFN) variants with truncated histidine tags were prepared and conjugation was examined using a bis-alkylation reagent, PEG(10kDa)-mono-sulfone 3. A histidine tag comprised of two histidines separated by a glycine (His2-tag) underwent PEGylation. Two more IFN variants were then prepared with the His2-tag engineered at different locations in IFN. Another IFN variant was prepared with the His-tag introduced in an α-helix, and required three contiguous histidines to ensure that two histidine residues in the correct conformation would be available for conjugation. Since histidine is a natural amino acid, routine methods of site-directed mutagenesis were used to generate the IFN variants from E. coli in soluble form at titres comparable to native IFN. PEGylation conversions ranged from 28-39%. A single step purification process gave essentially the pure PEG-IFN variant (>97% by RP-HPLC) in high recovery with isolated yields ranging from 21-33%. The level of retained bioactivity was strongly dependent on the site of PEG conjugation. The highest biological activity of 74% was retained for the PEG10-106(HGHG)-IFN variant which is unprecedented for a PEGylated IFN. The His2-tag at 106(HGHG)-IFN is engineered at the flexible loop most distant from IFN interaction with its dimeric receptor. The biological activity for the PEG10-5(HGH)-IFN variant was determined to be 17% which is comparable to other PEGylated IFN conjugates achieved at or near the N-terminus that have been previously described. The lowest retained activity (10%) was reported for PEG10-120(HHH)-IFN which was prepared as a negative control targeting a IFN site thought to be involved in receptor binding. The presence of two histidines as a His2-tag to generate a site-selective target for bis-alkylating PEGylation is a feasible approach for achieving site-selective PEGylation. The use of a His2-tag to strategically engineer a conjugation site in a protein location can result in maximising the retention of the biological activity following protein modification.

Comparing mutagenesis and simulations as tools for identifying functionally important sequence changes for protein thermal adaptation

Comparative studies of orthologous proteins of species evolved at different temperatures have revealed consistent patterns of temperature-related variation in thermal stabilities of structure and function. However, the precise mechanisms by which interspecific variations in sequence foster these adaptive changes remain largely unknown. Here, we compare orthologs of cytosolic malate dehydrogenase (cMDH) from marine molluscs adapted to temperatures ranging from -1.9 °C (Antarctica) to ∼55 °C (South China coast) and show how amino acid usage in different regions of the enzyme (surface, intermediate depth, and protein core) varies with adaptation temperature. This eukaryotic enzyme follows some but not all of the rules established in comparisons of archaeal and bacterial proteins. To link the effects of specific amino acid substitutions with adaptive variations in enzyme thermal stability, we combined site-directed mutagenesis (SDM) and in vitro protein experimentation with in silico mutagenesis using molecular dynamics simulation (MDS) techniques. SDM and MDS methods generally but not invariably yielded common effects on protein stability. MDS analysis is shown to provide insights into how specific amino acid substitutions affect the conformational flexibilities of mobile regions (MRs) of the enzyme that are essential for binding and catalysis. Whereas these substitutions invariably lie outside of the MRs, they effectively transmit their flexibility-modulating effects to the MRs through linked interactions among surface residues. This discovery illustrates that regions of the protein surface lying outside of the site of catalysis can help establish an enzyme's thermal responses and foster evolutionary adaptation of function.

Extended structure of pleiotropic DNA repair-promoting protein PprA from Deinococcus radiodurans.

Pleiotropic protein promoting DNA repair A (PprA) is a key protein facilitating the extreme radiation resistance of Deinococcus radiodurans. PprA is a unique protein to the genus Deinococcus and exists as an oligomer ranging from a tetramer to an ∼100-mer depending on protein concentrations. Here, the X-ray crystal structure of PprA was determined to clarify how PprA confers radiation resistance. The tertiary structure of dimeric PprA was elucidated by using mutants obtained with random and site-directed mutagenesis methods (W183R and A139R); these mutants have disabled DNA binding and polymerization functions. Because the mutant A139R and W183R proteins have dimeric assemblies with 2 different interfaces (Interfaces 1 and 2), the linear and oligomerized PprA model was constructed as a left-handed face-to-face periodic screw structure. In addition, the linear structure in solution was confirmed by small-angle scattering experiments. The site-directed mutational analysis identified essential basic amino acids for DNA binding. These analytical data support the hypothesis that a complex assembly of PprA molecules, which are extended and have a screw structure, surrounds and stretches the DNA strand, acting as a novel guide to colocalize the DNA strands for efficient DNA repairs.-Adachi, M., Shimizu, R., Shibazaki, C., Satoh, K., Fujiwara, S., Arai, S., Narumi, I., Kuroki, R. Extended structure of pleiotropic DNA repair-promoting protein PprA from Deinococcus radiodurans.

T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis.

The assembly of DNA fragments with homologous arms is becoming popular in routine cloning. For an in vitro assembly reaction, a DNA polymerase is often used either alone for its 3′-5′ exonuclease activity or together with a 5′-3′ exonuclease for its DNA polymerase activity. Here, we present a ‘T5 exonuclease DNA assembly’ (TEDA) method that only uses a 5′-3′ exonuclease. DNA fragments with short homologous ends were treated by T5 exonuclease and then transformed into Escherichia coli to produce clone colonies. The cloning efficiency was similar to that of the commercial In-Fusion method employing a proprietary DNA polymerase, but higher than that of the Gibson method utilizing T5 exonuclease, Phusion DNA polymerase, and DNA ligase. It also assembled multiple DNA fragments and did simultaneous site-directed mutagenesis at multiple sites. The reaction mixture was simple, and each reaction used 0.04 U of T5 exonuclease that cost 0.25 US cents. The simplicity, cost effectiveness, and cloning efficiency should promote its routine use, especially for labs with a budget constraint. TEDA may trigger further development of DNA assembly methods that employ single exonucleases.

Identification and Mutational Analysis of Escherichia coli Sorbitol-Enhanced Glucose-Repressed srlA Promoter Expressed in LB Medium by Using Homologous Recombination and One-Round PCR Products.

Escherichia coli has been used for recombinant protein production for many years. However, no native E. coli promoters have been found for constitutive expression in LB medium. To obtain high-expression E. coli promoters active in LB medium, we inserted various promoter regions upstream of eEmRFP that encodes a red fluorescent protein. Among the selected promoters, only colonies of srlA promoter transformants turned red on LB plate. srlA is a gene that regulates sorbitol utilization. The addition of sorbitol enhanced eEmRFP expression but glucose and other sugars repressed, indicating that srlAp is a sorbitol-enhanced glucose-repressed promoter. To analyze the srlAp sequence, a novel site-directed mutagenesis method was developed. Since we demonstrated that homologous recombination in E. coli could occur between 12-bp sequences, 12-bp overlapping sequences were attached to the set of primers that were designed to produce a full-length plasmid, denoted "one-round PCR product." Using this method, we identified that the srlA promoter region was 100bp. Further, the sequence adjacent to the start codon was found to be essential for high expression, suggesting that the traditionally used restriction enzyme sites for cloning in the promoter region have hindered expression. The srlA-driven expression system and DNA manipulation with one-round PCR products are useful tools in E. coli genetic engineering.

Crystal structure of mutant carboxypeptidase T from Thermoactinomyces vulgaris with an implanted S1' subsite from pancreatic carboxypeptidase B.

A site-directed mutagenesis method has been used to obtain the G215S/A251G/T257A/D260G/T262D mutant of carboxypeptidase T from Thermoactinomyces vulgaris (CPT), in which the amino-acid residues of the S1' subsite are substituted by the corresponding residues from pancreatic carboxypeptidase B (CPB). It was shown that the mutant enzyme retained the broad, mainly hydrophobic selectivity of wild-type CPT. The mutant containing the implanted CPB S1' subsite was crystallized and its three-dimensional structure was determined at 1.29 Å resolution by X-ray crystallography. A comparison of the three-dimensional structures of CPT, the G215S/A251G/T257A/D260G/T262D CPT mutant and CPB showed that the S1' subsite of CPT has not been distorted by the mutagenesis and adequately reproduces the structure of the CPB S1' subsite. The CPB-like mutant differs from CPB in substrate selectivity owing to differences between the two enzymes outside the S1' subsite. Moreover, the difference in substrate specificity between the enzymes was shown to be affected by residues other than those that directly contact the substrate.

Gene cloning and seamless site-directed mutagenesis using single-strand annealing (SSA)

The full length of interested genes can be usually cloned by assembling exons or RACE products through overlap PCR. However, the procedure requires multiple PCR steps, which are prone to random mutagenesis. Here, we present a novel SSA-based method for gene cloning and seamless site-directed mutagenesis. We firstly cloned the full-length coding sequence of Cashmere goat (Capra hircus) Hoxc13 gene by assembling exons amplified from genomic DNA. Secondly, we created a Hoxc13 loss-function mutant seamlessly and further illustrated that direct repeat length of 25bp is enough to trigger the SSA repair in routine E. coli strains including DH5α, Trans1t1, JM109, and Top10. Moreover, we cloned another full-length mutant of Foxn1 gene from Cashmere goat cDNA using further shortened direct repeats of 19bp. In summary, our study provided an alternative method to overcome the difficulties during overlap PCR in some particular cases for gene cloning.

Regulation of miR-34b/c-targeted gene expression program by SUMOylation.

The miR-34 family of microRNAs suppresses the expression of proteins involved in pluripotency and oncogenesis. miR-34 expression is frequently reduced in cancers; however, the regulation of their expression is not well understood. We used genome-wide miRNA profiling and mechanistic analysis to show that SUMOylation regulates miR-34b/c expression, which impacts the expression of c-Myc and other tested miR-34 targets. We used site-directed mutagenesis and other methods to show that protein kinase B (also known as Akt) phosphorylation of FOXO3a plays an important role in SUMOylation-dependent expression of miR-34b/c. This study reveals how the miR-34-targeted gene expression program is regulated by SUMOylation and shows that SUMOylation need not regulate target proteins through direct modification, but instead can act through the expression of their targeting miRNAs.

Combinatorial multispectral, thermodynamics, docking and site-directed mutagenesis reveal the cognitive characteristics of honey bee chemosensory protein to plant semiochemical

In the chemoreceptive system of insects, there are always some soluble binding proteins, such as some antennal-specific chemosensory proteins (CSPs), which are abundantly distributed in the chemosensory sensillar lymph. The antennal-specific CSPs usually have strong capability to bind diverse semiochemicals, while the detailed interaction between CSPs and the semiochemicals remain unclear. Here, by means of the combinatorial multispectral, thermodynamics, docking and site-directed mutagenesis, we detailedly interpreted a binding interaction between a plant semiochemical β-ionone and antennal-specific CSP1 from the worker honey bee. Thermodynamic parameters (ΔH < 0, ΔS > 0) indicate that the interaction is mainly driven by hydrophobic forces and electrostatic interactions. Docking prediction results showed that there are two key amino acids, Phe44 and Gln63, may be involved in the interacting process of CSP1 to β-ionone. In order to confirm the two key amino acids, site-directed mutagenesis were performed and the binding constant (KA) for two CSP1 mutant proteins was reduced by 60.82% and 46.80% compared to wild-type CSP1. The thermodynamic analysis of mutant proteins furtherly verified that Phe44 maintained an electrostatic interaction and Gln63 contributes hydrophobic and electrostatic forces. Our investigation initially elucidates the physicochemical mechanism of the interaction between antennal-special CSPs in insects including bees to plant semiochemicals, as well as the development of twice thermodynamic analysis (wild type and mutant proteins) combined with multispectral and site-directed mutagenesis methods.

Research on homology modeling, molecular docking of the cellulase and highly expression of the key enzyme (Bgl) in Pichia pastoris

Cellulose is the most abundant and renewable biological resource on earth. As nonrenewable resources are becoming scarce, cellulose is expected to become a major raw material for food, energy, fuel and other products. 1,4-β-glucosidase (Bgl), as a kind of cellulose, can be degraded cellulose into industrial available glucose. In this study, we constructed mutants of Bgl with enhanced activity based on homology modeling, molecular docking, and the site-directed mutagenesis of target residues to modify spatial positions, steric hindrances, or hydrophilicity/hydrophobicity. On the basis of the high-activity mutations were got (N347S and G235 M) by using site-directed mutagenesis and screening methods and introduced in the Pichia pastoris expression system, the enzymatic properties of mutant enzymes were analysed. Assays of the activity of the purified Bgl revealed that the two mutants exhibited increased activity. The pPICZαA-G235 M and pPICZαA-N347S mutants exhibited a >33.4% and 44.8% increase in specific activity respectively, with similar pH, temperature and metal ion requirements, compared to wild-type Bgl. These findings would be good foundation for improving production properties of Bgl in the future.

Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis.

Engineering proteins for designer functions and biotechnological applications almost invariably requires (or at least benefits from) multiple mutations to non-contiguous residues. Several methods for multiple site-directed mutagenesis exist, but there remains a need for fast and simple methods to efficiently introduce such mutations – particularly for generating large, high quality libraries for directed evolution. Here, we present Darwin Assembly, which can deliver high quality libraries of >108 transformants, targeting multiple (>10) distal sites with minimal wild-type contamination (<0.25% of total population) and which takes a single working day from purified plasmid to library transformation. We demonstrate its efficacy with whole gene codon reassignment of chloramphenicol acetyl transferase, mutating 19 codons in a single reaction in KOD DNA polymerase and generating high quality, multiple-site libraries in T7 RNA polymerase and Tgo DNA polymerase. Darwin Assembly uses commercially available enzymes, can be readily automated, and offers a cost-effective route to highly complex and customizable library generation.

Efficient strategy for introducing large and multiple changes in plasmid DNA

While the QuikChange site-directed mutagenesis method and its later modifications are extremely useful and simple, they suffer from several drawbacks. Here, we propose a new method, named LFEAP mutagenesis (Ligation of Fragment Ends After PCR) for creating various mutations in plasmid by leveraging three existing concepts: inverse PCR, single primer PCR, and sticky-end assembly. The first inverse PCR on the target plasmid yielded linearized DNA fragments with mutagenic ends, and a second single primer PCR resulted in complementary single-stranded DNA fragments with the addition of overhangs at the 5′ end of each strand. The resulting single strands were then annealed to produce double-stranded DNA with free 5′ single-stranded DNA tails. These products with compatible sticky ends were efficiently assembled into a circular, mutagenized plasmid. With this strategy, multiple simultaneous changes (up to 15) and mutations in large plasmids (up to 50 kb) were achieved with high efficiency and fidelity. LFEAP mutagenesis is a versatile method that offers significant advantages for introducing large and multiple changes in plasmid DNA.

Role of His-His interaction in Ser474-His475-Tyr476 sequence of chondroitinase ABC I in the enzyme activity and stability

Despite clinical importance of chondroitinase ABC I, its application has been limited due to thermal instability as reported in the literature. There are various approaches to improve thermal stability of enzymes, among them, His-His interactions are believed generally as an effective means. In the present study and for preparing a His-His interaction, various mutations in the sequence of Ser474-His475-Tyr476 at catalytic domain of the enzyme were performed using site directed mutagenesis method. The effect of these mutations on activity, stability and structural features of cABC I was assessed. The study showed that establishment of His475-His476 pair in cABC I, did not improve thermal stability of the enzyme and inactivated it. The study also revealed the existence a hydrogen bond network in the central domain of the enzyme with a specific role for tyrosine 476. In this network, replacement of His475 with Ala and Try476 with His and Ala, deactivated and destabilized the enzyme; confirming their importance in the enzyme catalysis and stability. Also, it was found that Tyr476 has some important role in substrate binding, an issue which should be more investigated.

Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods

The Cys-His bridge as electron transfer conduit in the enzymatic catalysis of nitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti 2011 (SmNir) was evaluated by site-directed mutagenesis, steady state kinetic studies, UV–vis and EPR spectroscopic measurements as well as computational calculations. The kinetic, structural and spectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) SmNir variants were compared with the wild type enzyme. Molecular properties of H171D and C172D indicate that these point mutations have not visible effects on the quaternary structure of SmNir. Both variants are catalytically incompetent using the physiological electron donor pseudoazurin, though C172D presents catalytic activity with the artificial electron donor methyl viologen (kcat=3.9(4) s−1) lower than that of wt SmNir (kcat=240(50) s−1). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the Nδ1H…OC hydrogen bond that partially shortcuts the T1–T2 bridging Cys-His covalent pathway. The role of the Nδ1H…OC hydrogen bond in the pH-dependent catalytic activity of wt SmNir is also analyzed by monitoring the T1 and T2 oxidation states at the end of the catalytic reaction of wt SmNir at pH6 and 10 by UV–vis and EPR spectroscopies. These data provide insight into how changes in Cys-His bridge interrupts the electron transfer between T1 and T2 and how the pH-dependent catalytic activity of the enzyme are related to pH-dependent structural modifications of the T1–T2 bridging chemical pathway.

The single nucleotide polymorphism site of aquaporin-4 gene in patients with neuromyelitis optica.

The single nucleotide polymorphism (SNP) site within the aquaporin (AQP)-4 gene exons and its possible role in the pathogenesis of neuromyelitis optica (NMO) were studied. From March 2010 to June 2012, 72 patients with NMO from Xiangyang No. 1 People's Hospital, Hubei University of Medicine were enrolled in the NMO group. At the same time, 80 patients with multiple sclerosis (MS) were enrolled in our study as the MS group. Blood samples were collected and DNA was extracted for analysis of SNP sites of AQP4 gene. Specific site-directed mutagenesis method was used for site-directed mutagenesis on plasmid enhanced green fluorescence protein carrying AQP4 gene. Mutant plasmids were constructed and used for transfecting cell lines. The differences of anti-AQP4 antibody level in the cell line were analyzed. The possible correlation between AQP4 gene SNP sites and the pathogenesis of NMO were analyzed. In the NMO group, 6 SNP sites in AQP4 gene were located in exons 2 and 5. These included R108T, I110N, E280R, D281R, P295R and E317M. There was no SNP site in exons 1, 3 and 4. In the MS group, no SNP site was found in AQP4 gene. R108T, I110N, R108T/I110N, E280R/D281R, P295R and E317M cell lines were constructed in the NMO group, and anti-AQP4 antibody in the serum was compared between R108T/I110N, E280R/D281R and E317M cell lines and the original HEK293T cell line. The difference was statistically significant (P<0.05). The positive rate of anti-AQP4 antibody titer in serum was compared between R108T, I110N, R108T/I110N, E280R/D281R, P295R and E317M cell lines in the NMO group and the original cell line in the MS group. In conclusion, SNP sites in AQP4 gene in patients with NMO may lead to some conformational changes in AQP4 protein. This affects the antigenicity of AQP4 protein. The different intensity of antigen-antibody reaction may cause the differences of titer observed between the different mutant cell lines.

Base-resolution stratification of cancer mutations using functional variomics.

A complete understanding of human cancer variants requires new methods to systematically and efficiently assess the functional effects of genomic mutations at a large scale. Here, we describe a set of tools to rapidly clone and stratify thousands of cancer mutations at base resolution. This protocol provides a massively parallel pipeline to achieve high stringency and throughput. The approach includes high-throughput generation of mutant clones by Gateway, confirmation of variant identity by barcoding and next-generation sequencing, and stratification of cancer variants by multiplexed interaction profiling. Compared with alternative site-directed mutagenesis methods, our protocol requires less sequencing effort and enables robust statistical calling of allele-specific effects. To ensure the precision of variant interaction profiling, we further describe two complementary methods-a high-throughput enhanced yeast two-hybrid (HT-eY2H) assay and a mammalian-cell-based Gaussia princeps luciferase protein-fragment complementation assay (GPCA). These independent assays with standard controls validate mutational interaction profiles with high quality. This protocol provides experimentally derived guidelines for classifying candidate cancer alleles emerging from whole-genome or whole-exome sequencing projects as 'drivers' or 'passengers'. For ∼100 genomic mutations, the protocol-including target primer design, variant library construction, and sequence verification-can be completed within as little as 2-3 weeks, and cancer variant stratification can be completed within 2 weeks.

Enhanced Mutant Screening in One-step PCR-based Multiple Site-directed Plasmid Mutagenesis by Introduction of Silent Restriction Sites for Structural and Functional Study of Proteins

Site-directed mutagenesis (SDM) has been widely used for studying the structure and function of proteins. A one-step polymerase chain reaction (PCR)-based multiple site-directed plasmid mutagenesis method with extended non-overlapping sequence at the 3′ end of the primer increases the PCR amplification efficiency and the capacity of multi-site mutagenesis. Here, we introduced silent restriction sites in the primers used in this PCR-based SDM method by utilizing SDM-Assist software to generate mutants of Helicobacter pylori neutrophil-activating protein (HP-NAP), whose gene has low GC content. The HP-NAP mutants were efficiently generated by this modified mutagenesis method and quickly identified by a simple restriction digest due to the presence of the silent restriction site. This modified PCR-based SDM method with the introduction of a silent restriction site on the primer is efficient for generation and identification of mutations in the gene of interest.