Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies

Edelheit, Oded; Hanukoglu, Aaron; Hanukoglu, Israel

doi:10.1186/1472-6750-9-61

Cited by 309 publications

(240 citation statements)

References 11 publications

(12 reference statements)

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“…Expression vectors were obtained 20 and Chinese hamster ovary (CHO) cells were transfected as described in the Online Supplementary Data. All experiments were performed 2 days after transfection.…”

Section: Construction Of the Expression Vectors Mutagenesis And Tranmentioning

confidence: 99%

Cytoskeletal perturbation leads to platelet dysfunction and thrombocytopenia in variant forms of Glanzmann thrombasthenia

et al. 2015

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Several patients have been reported to have variant dominant forms of Glanzmann thrombasthenia, associated with macrothrombocytopenia and caused by gain-of-function mutations of ITGB3 or ITGA2B leading to reduced surface expression and constitutive activation of integrin α IIb β 3 . The mechanisms leading to a bleeding phenotype of these patients have never been addressed. The aim of this study was to unravel the mechanism by which ITGB3 mutations causing activation of α IIb β 3 lead to platelet dysfunction and macrothrombocytopenia. Using platelets from two patients carrying the β 3 del647-686 mutation and Chinese hamster ovary cells expressing different α IIb β 3 -activating mutations, we showed that reduced surface expression of α IIb β 3 is due to receptor internalization. Moreover, we demonstrated that permanent triggering of α IIb β 3 -mediated outside-in signaling causes an impairment of cytoskeletal reorganization arresting actin turnover at the stage of polymerization. The induction of actin polymerization by jasplakinolide, a natural toxin that promotes actin nucleation and prevents depolymerization of stress fibers, in control platelets produced an impairment of platelet function similar to that of patients with variant forms of dominant Glanzmann thrombasthenia. del647-686β 3 -transduced murine megakaryocytes generated proplatelets with a reduced number of large tips and asymmetric barbell-proplatelets, suggesting that impaired cytoskeletal rearrangement is the cause of macrothrombocytopenia. These data show that impaired cytoskeletal remodeling caused by a constitutively activated α IIb β 3 is the main effector of platelet dysfunction and macrothrombocytopenia, and thus of bleeding, in variant forms of dominant Glanzmann thrombasthenia. Cytoskeletal perturbation leads to platelet dysfunction and thrombocytopenia in variant forms of Glanzmann thrombasthenia

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Section: Construction Of the Expression Vectors Mutagenesis And Tranmentioning

confidence: 99%

Cytoskeletal perturbation leads to platelet dysfunction and thrombocytopenia in variant forms of Glanzmann thrombasthenia

et al. 2015

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“…SDM symbolized as the prime method to engineer proteins and for changing enzyme substrate selectivity (Edelheit et al, 2009). The importance of SDM in protein engineering by changing a specific amino acid and creating a modified sequence was used in the UGTs to change a specific amino acid and studied its effects on the enzyme substrate specificity.…”

Section: Cloning Of Recombinant Plasmids After Site Directed Mutagenementioning

confidence: 99%

Cloning and Site Directed Mutagenesis of UGT76E1 Leads to Changed Substrate Activity in Arabidopsis thalian

Majeed¹,

Zia²,

Yang³

et al. 2015

IJAB

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Glycosyltransferases are ubiquitous enzymes play vital role in numerous aspects of living organisms and are involved in secondary metabolism. Also known as uridine diphosphate (UDP) glycosyltransferases (UGTs) are engaged in detoxification in living organisms. UGT76E1 is a member of 76E family of UGT comprising of 453 amino acids. The gene for glycosyltransferase, UGT76E1 from Arabidopsis thaliana was expressed and purified through affinity chromatography.Site directed mutagenesis was performed to change hydrophilic threonine (T) into hydrophobic alanine (A) at 134 position. Afterwards the activity of mutant enzyme was analyzed through mass spectrometry. Novobiocin and kaempherol were used as acceptor moleculesfor activity tests. After mutagenesis the mutant UGT76E1T134A lost its activity with its donor sugar UDP glucose, as no peak was observed for the required products glc-novobiocin and glc-kaempherol at 773 and 447 in mass spectrum respectively. The mutant did not attainany new activity with UDP rhamnose also. Complete loss of activity of UGT76E1T134A with UDP glucose as donor sugar suggested for the presence of significant peptides at the site of mutation.The current results showed that glycosyltransferases can be modified to use different substrates by site directed mutagenesis. This UGT enzyme modification could open new horizon in the development of new drugs for cancer treatment.

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“…3. Extract and purify the sRNA expression phagemid and helper plasmid using a DNA extraction kit or similar method 12 . 4.…”

Section: Production and Harvest Of M13-packaged Phagemid Stocksmentioning

confidence: 99%

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in <em>E. coli</em>

Bernheim

Libis

Lindner

et al. 2016

JoVE

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RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISCexpressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

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Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies

Cited by 309 publications

References 11 publications

Cytoskeletal perturbation leads to platelet dysfunction and thrombocytopenia in variant forms of Glanzmann thrombasthenia

Cytoskeletal perturbation leads to platelet dysfunction and thrombocytopenia in variant forms of Glanzmann thrombasthenia

Cloning and Site Directed Mutagenesis of UGT76E1 Leads to Changed Substrate Activity in Arabidopsis thalian

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in <em>E. coli</em>

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