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Protein engineers are working to develop engineered proteases, which have capability to proceed more competently at low temperature and alkaline pH.

Mesophilic subtilis in proteases from B. These subtilisin-like proteases show 9. Mutations in more than amino acid s of subtilisin have been reported. Subtilisin BPN, subtilisin E and Savinase are most mutagenized proteases used industrial processes Protein engineering and cloning techniques have made possible to produce commercial proteases with required characters of pH and temperature activity and stability.

It has also modified the bacterial species to produce large quantities of enzymes under different stress conditions 46 , Amylases are employed in various industries to multiply functions for example it is used in food industry for softening bread, adjusting flour for liquefaction and scarification of starch as well as juice treatment. In detergent and paper industry, these enzymes are frequently used to eliminate starch stains and de-inking For the production of certain food and industrial products starch is converted into bioethanol or into food ingredients like fructose, glucose and organic acid s in microbial fermenters, requiring biocatalysts such amylase for the liquefaction and scarification.

Thus to improve the activity and stability of amylases at harsh conditions both protein engineering and DNA recombinant technology are being frequently used. Rice has been well reported as an instance for the production of industrial useful biocatalysts from raw material of agriculture Lipases are also used intensively by food and detergent industries such as for lipid stain removal, chees flavor, dough stability and as contaminants controller in paper and pulp industry.

For food processes toxicologically safe lipases are required which are obtained from Candid arugose. Different commercial isoforms of lipases are produced by DNA shuffling, computer modeling and protein engineering Later on a comprehensive study was accomplished on mutagenesis and protein engineering to enhance the catalysis of microbial lipases Applications referring to remediation of polluted environments oxygenases, laccases and peroxidases are three major classes of enzymes, which have significant role in environmental applications for biodegradation of organic and toxic pollutants.

But mostly, these enzymes face problems like enzyme denaturation by toxic compounds, inhibition of ES enzyme-substrate complex and low catalytic activity. Scientists have done intensive work to overcome these problems by developing engineered enzymes by recombinant technology and rational enzyme design Medical and clinical applications: Protein engineering has vast number of applications in the area of therapeutics. Formerly protein engineering is accomplished to achieve second generation recombinant protein having considerable properties in medical and clinical applications Mutation, DNA shuffling and recombinant DNA approach were used in protein engineering to get superior results of therapeutic protein Afterward up-gradation in protein engineering led to fabrication of secreted therapeutic proteins, namely, interferon, insulin, etc.

Up-gradation of therapeutics for combating against cancer is the major area of interest in protein engineering. One of latent treatment recommended for cancer is pre-targeted immunotherapy in which radiation toxicity is noticed to be minimized.

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By using protein engineering, the application of this pre-targeted immunotherapy was anticipated to be a competent treatment for cancer Up-gradation in recombinant DNA technology and protein engineering facilitates the synthesis of novel antibodies that can be successfully applied as anti-cancer drugs. These distinctive antibodies are engineered such a manner that they specifically recognize and strongly associated with their cancerous antigenic markers and assist in eliminating the cancerous cell with greater precision.

Development in protein engineering leads to some of its other noteworthy medical applications. One of them is protein cationization technique, which assists in development of future therapeutics Tissue regeneration and polymer based drug delivery system was another major target of protein engineering Targeted drug delivery remains the important feature of a novel biopharmaceutical to attain successful therapies. Functional proteins and peptides are engineered with an efficient carrier for sufficient and targeted delivery of drug in this apprehension.

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  5. Certainly, health care can be more operational if the diagnosis is speedy, accurate and perceptive. Nearly genetic disorders have been reported so far.

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    The majority of human contain a few genes without any sign of disease and many of them are accountable for susceptibility, however molecular basis of majority of these diseases is still unclear. Successful efforts have been made in last more than three decades in sighting into diagnosing genetic disorders prior to embryonic implantation in humans and credited a lot of merit. Beadle and Tatum anticipated one gene-one hypothesis, was condemned later has shown that certain genes consequence in dozens of proteins 64 , probably get produced either in traces with a very short half-life, splitted, chemically changed or the fragments of different genes may be reorganized.

    For that specific reason, under such physiological conditions gene analysis is not suitable in clinical diagnosis of the proteins and eventually proteomics desires the characterization of certain proteins that are key agents of a cell and gene products.

    These agents straightforward contribute to the drug development as all drugs are directed against proteins, except a few, get in the way in DNA replication in cancer cells and RNA in AIDS virus multiplicity. Thus, advancements in protein detection and characterization protocols would assist in diagnosing diseases with accuracy and sensitivity.

    Hereafter, up-gradation in protein nano-technologies having been carried out in recent years, is comprehensively updated here. It is fairly noteworthy to monitor the protein concentration in a biological sample prior to investing for its practical biological activity. The accurate estimation of less abundant protein is the prime challenge, having been overcome by evolution of nano-technology Fluorometric assay 68 , ELISA 69 , radioimmunoassay 69 and immunofluorosence 69 measurement tools are evolved to quantify the proteins in nano quantity and even less, although, except spectrofluorometric 68 technique, those are multi-step, difficult and rather time-consuming techniques.

    Protein engineering in nano-biotechnology: The applications of protein engineering in nano-biotechnology are moving ahead with the time.

    Directed Evolution of Enzymes

    Nanotechnology was not receiving substantial credit for their difficult synthesis and assembly in functional systems. Then after, a phase came with the studies on biomolecular structural organizations revealing their hierarchical arrangements from nano to macro levels. Proteins, lipids and carbohydrates are the biological macromolecules, being used for biosynthesis of tissues under synchronized gene expression s. Proteins are the most noteworthy amongst them as they are the structural constituents during tissue formation and aid to the transport and arrangement of building blocks and accessories.

    Therefore proteins are the major focus for nano-technological systems in their synchronized synthesis and assemblage. The combinatorial tools of biology used in protein engineering such as the technologies of bacterial cell surface display and phage display also get their applications in nanobiotechnology to monitor selectively binding polypeptide sequences to inorganic surfaces. Individual clones, likely to be specific in their binding to an inorganic material surface are principally revealed through stepwise washings of phages or cells in the biological method named as bio-panning.

    Sequencing of these clones is performed in view of obtaining the amino acid sequences of these polypeptides, purposely bind to semi-metal oxides and other nano-technology surfaces. Nano-biotechnology did extremely well further through another technique employing Genetically Engineered Proteins for inorganics i.

    Subsequently, a number of specific peptides, being bound to certain surfaces like quartz and gold, have been selected and characterized 70 , Besides, computational methods were combined with experimental approaches in view of better engineering the binding of peptides followed by accurate assembly of nano-technology systems revealing superior function specific peptides that can be used in therapeutics, tissue engineering and nano-technologies employing biological, organic and inorganic materials Protein engineered peptides are employed in biosensors, used as molecular motors and transducers, in the generation of biocompatible nano-materials.

    Bioinformatics analyses have also great impact in this emerging field of protein engineering Amyloid fibrils are also attractive application of protein engineering in the construction of nano wires as they provide as the templates. In fact, this is a characteristic of many of the proteins that they figure an organized aggregate of fibrils, namely, amyloid fibrils. This salient feature of well-organized non-covalent aggregate formation ability of amyloid fibrils directs their use in nano-technology with self-assembly and organization of small molecules being quite specific and vital Pioneering proteins recognized as affibody binding proteins, being of non-immunoglobulin Ig origin have been developed employing protein engineering techniques.

    They have high affinity and thus are potentially considered in diagnostics, viral targeting, bio-separation and tumor imaging as well 73 , For development of novel biosensors for analytical diagnosis, insertional protein engineering has been noticed to immerge during a decade 1 , The amino acid succession and organization in a protein affects its conformation as well as function.

    Consequently, the capability to transform the sequence and thus the structure and activity, of entity proteins in a methodical fashion, explore many opportunities, both scientifically and for exploitation in bio-catalysis. Modern techniques of synthetic biology, whereby increasingly large sequences of DNA can be synthesized de novo , allow an incomparable ability to engineer proteins possessing novel functions.

    Enzymologists differentiate binding K d and catalytic k cat stages. In a similar manner, judicious approaches have blended design for binding, specificity and active site modeling with more empirical methods of classical directed evolution DE for improving k cat where natural evolution rarely pursues the highest values , principally with respect to residues distant from the active site and where the functional linkages supporting enzyme dynamics are both unknown and hard to predict. The aim of this overview is to bring to light some of the approaches, being developed to allow using directed evolution for improving enzyme characteristics, often noticeably.

    It has been registered that directed evolution varies in a various ways from natural evolution, including in picky the accessible mechanisms and the potential selection pressures. Therefore, it is hereby firmly focused on opportunities afforded by techniques, which enable protein engineer or enzymologist to map sequence to structure and activity in silico , as an effective ways of modeling and thus exploring protein landscapes. As identified landscapes may be assessed and rational about as a whole, concurrently, this offers opportunities for protein improvement not readily available to natural evolution on rapid timescales.

    Intelligent landscape triangulation, experienced by sequence-activity interactions and joined to the promising techniques of synthetic biology, offers scope for the development of novel biocatalysts that are both extremely dynamic and strong. Further, for gene expression analysis, zinc finger protein engineering is becoming fascinating for molecular biologists. Afterward a three-finger protein was effectively engineered to study the expression of an oncogene in mouse cell line 75 , The understanding of gene regulation and structure and function of the human genome improved dramatically at the end of the 20th century.

    Conversely, the technologies for manipulating the genome have been slower to develop. For example, the arena of gene therapy has been focused on correcting genetic diseases and increasing tissue repair for more than four decades.

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    Though, with the exception of a few very low efficiency techniques, conformist genetic engineering approaches have only been competent to supplement auxiliary genes to cells. This has been a substantial complication to the clinical success of gene therapies and has also intended for severing inadvertent concerns in several cases.

    Week 10- Protein Engineering Lecture 10: Directed Evolution

    Consequently, technologies that make possible the defined modification of cellular genomes have diverse and notable implications in many facets of research and are noteworthy for translating the products of the Genomic Revolution into perceptible benefits for medicine and biotechnology. To address this requirement, in s, a task was embarked to expand technologies for engineering protein-DNA interactions with the rationale of generating custom tools competent of targeting any DNA sequence. The objective has been to let researchers to reach into genomes to specifically control, knock out, or replace any gene.

    To realize these aims, it has principally been focused on understanding and manipulating zinc finger proteins. Specifically, it is required to create a simple and straight forward method that enables unspecialized laboratories to engineer custom DNA-modifying proteins employing only defined modular components, a web-based usefulness and standard recombinant DNA technology.

    Two substantial challenges faced so far were i The development of zinc finger domains that target sequences not recognized by naturally occurring zinc finger proteins and ii Determining how individual zinc finger domains could be chained together as polydactyl proteins to identify exclusive locations within complex genomes.

    Various researchers have since employed this modular assembly technique to engineer artificial proteins and enzymes, which activate, repress or make definite changes to user-specified genes in human cells, plants and other organisms. Besides, they engineered certain novel techniques for externally regulating protein activity and delivery have been successfully developed 76 , as well as developed certain new approaches for the directed evolution of protein and enzyme function.

    However, in biofuel industry, to obtain biofuels from lignocellulosic materials, such cellulose enzymes are produced by protein engineering, which have improved catalytic activity and reduced the production costs of biofuels Protein cysteine modification, an approach of protein engineering, produces proteins with diverse functions 78 , The usage of proteins as therapeutics has a long history and is becoming ever more common in modern medicine.

    Despite the fact that number of protein-based drugs is growing every year, major problems still remain with their application. Among these complications are quick degradation and excretion from patients, consequently requiring recurrent dosing that in turn increases the chances for an immunological response as well as increasing the cost of therapy.

    One of the main strategies to improve these problems is to link a polyethylene glycol PEG group to the protein of interest.

    Enzymes high potential biocatalyst in our libraries

    This procedure called PEGylation has grown strongly in recent years occasioning in several approved drugs. Installing a single PEG chain at a definite site in a protein is quite challenging.