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Role of Engineered Proteins as Therapeutic Formulations
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Khushboo Gulati, Krishna Mohan Poluri
In contrast to random mutagenesis, focused mutagenesis involves generation of small libraries that can be readily screened. Smaller libraries are the result of mutations that are focused only to protein hot spot residues. Hot spots correspond to the residues which have phenomenal functional/structural role. Site saturation mutagenesis (SSM) is the most commonly employed focused mutagenesis method which tries all the 20 amino acids against the targeted amino acid in a single shot. The two most commonly employed SSM methods are: (1) site directed mutagenesis (SDM) that uses complementary primer pairs containing the mutation site (Matsumura and Rowe, 2005; Alcalde et al., 2006). (2) Overlap extension PCR that employs two pairs of complementary primers with two rounds of PCR to introduce the mutation at the targeted site (Gratz and Jose, 2008). Other focused mutagenesis methods such as cassette mutagenesis (Reidhaar-Olson and Sauer, 1988), sequence saturation mutagenesis (SeSAM) (Wong et al., 2008), single-primer reactions in parallel (SPRINP) (Edelheit et al., 2009), megaprimed and ligase-Free (Tseng et al., 2008), Ω-PCR (Chen et al., 2013), PFunkel (Firnberg and Ostermeier, 2012), ominchange (Dennig et al., 2011), OSCARR (Hidalgo et al., 2008), trimer-dimer mutagenesis (Gaytan et al., 2009), synthetic saturation mutagenesis (Patwardhan et al., 2009), Amber codon saturation mutagenesis have also been developed and are being employed for the creation of variant libraries (Shozen et al., 2012).
Förster Resonance Energy Transfer Microscopy for Monitoring Molecular Dynamics in Living Cells
Published in Guy Cox, Fundamentals of Fluorescence Imaging, 2019
Vinod Jyothikumar, Yuansheng Sun, Ammasi Periasamy
Today, a considerable number of crystal structures of GFP-like proteins are available, which can help to identify target residues for knowledge-based genetic engineering. The primer overlap extension PCR is a very robust technique to perform directed amino acid exchanges [33–35]. Oligonucleotide primers can be also designed to randomize a distinct residue so that any possible amino acid will be introduced in this position. The randomization of defined residues can be used, for instance, to verify quickly if a mutation picked up during screening of a random mutant library is indeed the optimal exchange in a certain position. Examples of successful applications of site-directed mutagenesis include the disruption of tetrameric associations or the generation of variants with strongly red-shifted emission [36–38].
Bacteriophage Scaffolds for Functional Assembly of Molecules and Nanomaterials
Published in Gilson Khang, Handbook of Intelligent Scaffolds for Tissue Engineering and Regenerative Medicine, 2017
Mi Hwa Oh, Jeong Heon Yu, Moon Young Yang, Yoon Sung Nam
Unlike a widely used phagemid system, the type 8 phage has the advantages of displaying inorganic crystals with defined length and width using homogenous display on pVIII. A type 8 phage display system has been constructed by engineering pVIII proteins to display selected octapeptides that were displayed at distances about 2.7 nm from each other. To construct a type 8 phage display system, a new cloning site between PstI and BamH1 at positions 1372 and 1381, respectively, was created in the M13KE phage vector, and the original PstI site at position 6246 was deleted by single point mutation. Overlap extension PCR was performed for site-directed mutagenesis. A double stranded DNA library was prepared and then cloned into the modified phage vector, named M13SK.29 Moreover, the type 8–3 phage system was used to create multifunctional phage displaying two different engineered peptides at both pIII and pVIII (Fig. 26.3).30
High yield expression, characterization, and biological activity of IFNα2-Tα1 fusion protein
Published in Preparative Biochemistry & Biotechnology, 2020
Muhammad Shahbaz Aslam, Iram Gull, Malik Siddique Mahmood, Muhammad Mudassir Iqbal, Zaigham Abbas, Imran Tipu, Aftab Ahmed, Muhammad Amin Athar
This study involves construction of IFNα2-Tα1 fusion gene coding sequence by overlap extension PCR. Overlap extension PCR is a variant of standard PCR which is considered as a simple and reliable way to construct a fusion-fragments with less than 2 kb size. The IFNα2-Tα1 fusion gene constructed in this study is composed of IFNα2 gene fused with Tα1 gene at its 3′ end with oligonucleotide encoding a linker peptide (Gly4Ser) inserted between two fragments. Further, this study involves expression optimization of IFNα2-Tα1 fusion protein in Escherichia coli under different conditions, purification of recombinant fusion protein, characterization and study of its biological activity. Our results demonstrated for the first time that IFNα2-Tα1 fusion protein could be over-produced in bacterial expression system with significantly higher yield using pET vector and modified terrific broth medium. The purified IFNα2-Tα1 fusion protein was biologically active with both IFNα2 & Tα1 activities and elevated anticancer activity.