Site‐Directed Mutagenesis by Polymerase Chain Reaction

Castorena‐Torres, Fabiola; Peñuelas-Urquides, Katia; León, MarioBermúdez de

doi:10.5772/66429

Cited by 3 publications

(3 citation statements)

References 22 publications

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“…However in our study, a more extended sequence containing 153 bp, has been successfully deleted using the site-directed mutagenesis. Suitability of the PCR condition and quality of enzymes is the crucial factor in site-directed mutagenesis (Castorena-Torres et al 2016). Moreover, a relatively small plasmid (~6000 bp) was used in the recent study.…”

Section: Discussionmentioning

confidence: 99%

“…The primer pairs used in directed mutagenesis were specifically designed to amplify the frontal sequence of the FLAG-tag (FP-Kex2-699) and to amplify the frontal sequence of the TMD (RP-Kex2-650), thus the TMD is not amplified. The two PCR mutagenesis primer sequences used the addition of restriction sites BamHI (5̍ -GGATCC-3̍ ) to facilitate the relegation process and increase the chances of successful site-directional mutagenesis (Castorena-Torres et al 2016). The optimization of PCR annealing temperature aims to optimize the PCR reaction process, by attaching primers to the target DNA and producing PCR products in optimal quantities (Kumari and Amaravathi 2016).…”

Section: Discussionmentioning

confidence: 99%

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Construction of Saccharomyces cerevisiae KEX2-650 gene expression vector and its introduction into Escherichia coli DH5?

ADIBAH

FUAD²,

Budiarti³

et al. 2022

Biodiversitas

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Abstract. Adibah DI, Fuad AM, Budiarti S, Pratiwi RD. 2022. Construction of Saccharomyces cerevisiae KEX2-650 gene expression vector and its introduction into Escherichia coli DH5?. Biodiversitas 23: 4289-4296. Kex2 (EC 3.4.21.61) is a serine protease that binds Ca2+ molecules and naturally found intracellularly as a transmembrane protein. Kex2 has a unique function, the conversion process of recombinant protein precursor into mature protein. Kex2 from Saccharomyces cerevisiae has similar functions also with furin in mammals (about 47% of sequence similarity). To gain advantage, extracellular Kex2 would be highly favorable for this process. This study aimed to construct recombinant Kex2 that could be produced extracellularly in Pichia pastoris host through pD902-KEX2-699 vector (synthetic) with FLAG-tag and 6 His-tag by removing most of C-terminal region, including transmembrane domain (TMD) from KEX2 gene sequence. Constructed KEX2 is the KEX2-650 variant with TMD deletion and cytoplasmic domain. The recombinant plasmid was constructed through site-directed mutagenesis using FP-Kex2-699 and RP-Kex2-650 primers, including the BamHI site for plasmid religation. PCR site-directed mutagenesis produces an amplicon DNA with an expected length of 5551 bp. After restriction (BamHI) and religation, the plasmid was reintroduced into Escherichia coli DH5? and obtained 16 colonies. Verification PCR target gene showed that clones number 9 produced an amplicon of expected length (646 bp). DNA sequencing analysis confirmed that TMD was removed from the gene construct to form the KEX2-650 construct.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Construction of Saccharomyces cerevisiae KEX2-650 gene expression vector and its introduction into Escherichia coli DH5?

ADIBAH

FUAD²,

Budiarti³

et al. 2022

Biodiversitas

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“…Previously established E2 mutants E2_152 and E2_158 were used to engineer CBU1910-E2 fusion constructs. , D381C is an E2 mutation that introduces 60 cysteines to the internal cavity of the nanoparticle, allowing for internal conjugation. , To introduce the D381C mutation to E2_158 and E2_152 via site directed mutagenesis (SDM) , the forward primer: 5′-/5Phos/GCCGATCGTTCGTTGCGGTGAAATCGTTGC-3′ and reverse primer: 5′-/5Phos/TTTTCGGCTATACGACCAATACCCAG-3′ were used. To introduce the DNA cut sites required for ligation to the N-terminus of E2 mutants, Nde1 and Nhe1 cut sites were introduced to the N-terminus DNA coding region and C-terminus DNA coding region, respectively, of CBU1910 using the forward primer: 5′-CATATGCACCATCACCATCACCATCCGCAGCAAGTCAAAGACATTCAG-3′ and reverse primer: 5′-GCTAGCTTAGCCGCCGGTTTCCGG-3′.…”

Section: Methodsmentioning

confidence: 99%

Engineering Protein Nanoparticles Functionalized with an Immunodominant Coxiella burnetii Antigen to Generate a Q Fever Vaccine

Ramirez,

Felgner,

Jain

et al. 2023

Bioconjugate Chem.

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Coxiella burnetii is the causative agent of Q fever, for which there is yet to be an FDA-approved vaccine. This bacterial pathogen has both extra- and intracellular stages in its life cycle, and therefore both a cell-mediated (i.e., T lymphocyte) and humoral (i.e., antibody) immune response are necessary for effective eradication of this pathogen. However, most proposed vaccines elicit strong responses to only one mechanism of adaptive immunity, and some can either cause reactogenicity or lack sufficient immunogenicity. In this work, we aim to apply a nanoparticle-based platform toward producing both antibody and T cell immune responses against C. burnetii. We investigated three approaches for conjugation of the immunodominant outer membrane protein antigen (CBU1910) to the E2 nanoparticle to obtain a consistent antigen orientation: direct genetic fusion, high affinity tris-NTA-Ni conjugation to polyhistidine-tagged CBU1910, and the SpyTag/SpyCatcher (ST/SC) system. Overall, we found that the ST/SC approach yielded nanoparticles loaded with the highest number of antigens while maintaining stability, enabling formulations that could simultaneously co-deliver the protein antigen (CBU1910) and adjuvant (CpG1826) on one nanoparticle (CBU1910-CpG-E2). Using protein microarray analyses, we found that after immunization, antigen-bound nanoparticle formulations elicited significantly higher antigen-specific IgG responses than soluble CBU1910 alone and produced more balanced IgG1/IgG2c ratios. Although T cell recall assays from these protein antigen formulations did not show significant increases in antigen-specific IFN-γ production compared to soluble CBU1910 alone, nanoparticles conjugated with a CD4 peptide epitope from CBU1910 generated elevated T cell responses in mice to both the CBU1910 peptide epitope and whole CBU1910 protein. These investigations highlight the feasibility of conjugating antigens to nanoparticles for tuning and improving both humoral- and cell-mediated adaptive immunity against C. burnetii.

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Role of cloning and modification of genes in pesticide decomposition

Sharma¹,

Shobana²

2023

Current Developments in Biotechnology and Bioengineering

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Site‐Directed Mutagenesis by Polymerase Chain Reaction

Cited by 3 publications

References 22 publications

Construction of Saccharomyces cerevisiae KEX2-650 gene expression vector and its introduction into Escherichia coli DH5?

Construction of Saccharomyces cerevisiae KEX2-650 gene expression vector and its introduction into Escherichia coli DH5?

Engineering Protein Nanoparticles Functionalized with an Immunodominant Coxiella burnetii Antigen to Generate a Q Fever Vaccine

Role of cloning and modification of genes in pesticide decomposition

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