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Proteins and proteomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
Other possibilities exist as well. For example, immunohistochemistry usually utilizes an antibody to one or more proteins of interest that are conjugated to enzymes yielding either luminescent or chromogenic signals that can be compared between samples, allowing for localization information. Another applicable technique is co-fractionation in sucrose (or other material) gradients using isopycnic centrifugation. While this technique does not prove co-localization of a compartment of known density and the protein of interest, it does increase the likelihood and is more amenable to large-scale studies. Finally, the gold-standard method of cellular localization is immunoelectron microscopy. This technique also uses an antibody to the protein of interest, along with classical electron microscopy techniques. The sample is prepared for normal electron microscopic examination, and then treated with an antibody to the protein of interest that is conjugated to an extremely electro-dense material, usually gold. This allows for the localization of both ultra-structural details as well as the protein of interest. Through another genetic engineering application known as site-directed mutagenesis, researchers can alter the protein sequence and hence its structure, cellular localization, and susceptibility to regulation. This technique even allows the incorporation of unnatural amino acids into proteins, using modified tRNAs, and may allow the rational design of new proteins with novel properties.
Protein Engineering and Bionanotechnology
Published in Anil Kumar Anal, Bionanotechnology, 2018
The rational design uses site-directed mutagenesis where the amino acid is injected into a target gene. Two common methods for site-directed mutagenesis are: (1) overlapped extension method and (2) whole plasmid single-round polymerase chain reaction (PCR). In overlap extension method, two primer pairs and short sequences of synthetic DNA complementary to gene of interest are involved in the first PCR wherein two separate PCRs are performed. First PCR uses four primers resulting in two double-stranded DNAs, which on denaturation and annealing produce two heteroduplexes with desired mutagenic codon in each strand. The overlapping in 3′ and 5′ ends of each heteroduplex is filled by DNA polymerase and then the mutagenic DNA is amplified by second PCR using a nonmutated primer. After DNA polymerase PCR takes place, the desired mutated plasmid without overlap in it breaks due to replication of strands template. In the whole plasmid single-round PCR method, two oligonucleotide primers with desired sequences and complementary to the opposite strands of double-stranded DNA plasmid template are extended using DNA polymerase. During PCR, both strands are replicated without displacing the primers. Circular mutated plasmid is obtained upon transformation of the circular nicked vector (Antikainen and Martin 2005).
Selection and Improvement of Industrial Organisms for Biotechnological Applications
Published in Nduka Okafor, Benedict C. Okeke, Modern Industrial Microbiology and Biotechnology, 2017
Nduka Okafor, Benedict C. Okeke
The outcome of the conventional mutations is random and unpredictable. Recombinant DNA technology and the use of synthetic DNA now make it possible to have mutations at specific sites on the genome of the organism in a technique known as Site-Directed Mutagenesis. The mutation is caused by in vitro changes directed at a specific site in a DNA molecule. The most common method involves use of a chemically synthesized oligonucleotide mutant which can hybridize with the DNA target molecule; the resulting mismatch-carrying DNA duplex may then be transfected into a bacterial cell line and the mutant strands recovered. The DNA of the specific gene to be mutated is isolated, and the sequence of bases in the gene determined. Certain predetermined bases are replaced and the ‘new’ gene is reinserted into the organism. Site-directed mutagenesis creates specific, well-defined mutations (i.e. specific changes in the protein product). It has helped to raise the industrial production of enzymes as well as to produce specific enzymes.
A comprehensive review on enzymatic degradation of the organophosphate pesticide malathion in the environment
Published in Journal of Environmental Science and Health, Part C, 2019
Smita S. Kumar, Pooja Ghosh, Sandeep K. Malyan, Jyoti Sharma, Vivek Kumar
The major hurdle in using malathion as a substrate lies in its poor rate of hydrolysis. The use of naturally occurring enzymes that hydrolyze organophosphates 40–2000 times faster than chemical hydrolysis, has provided the opportunity for the development of natural biodegradation strategies.108 It has been demonstrated that directed evolution can be used for the generation of different OPH variants so as to increase the efficiency of detoxification. On the other hand, its substrate specificity and stereoselectivity can be improved through site-directed mutagenesis.107 The OPH toolbox contains phosphotriesterases, paraoxonase 1 and organophosphorus hydrolases from the b-lactamase superfamily.114