Some Early Achievements of Protein Engineering,Lactamase,Serine,Cysteine,Dihyrofolate Reductase,Insulin,Lactose Permease,Gene,Lysozyme

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Protein and Enzymes Engineering Protein and Enzymes Engineering - Introduction Engineering of Macromolecules Basic Outline Protein Engineering vs Enzyme Engineering for a Biocatalyst Protein Engineering Rationale of Protein Enzyme Engineering Basic Assumptions for Protein Engineering Key Biocatalyst Properties that are Altered Through Protein Engineering Some Examples of Improved Kinetic Parameters of Enzymes Turnover Determined by k_cat Selectivity and Specificity Some Technologies Available for Enchancing Biocatalyst Characteristics Molecular Stability Catalytic Diversity Acquiring Knowledge for Protein Engineering Study of Protein Structure Three Dimensional Protein Modeling Perturbation Theory

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Your Ad Here	Some Early Achievements of Protein Engineering A number of proteins are known now, where efforts have been made to know the effects of site specific mutagenesis involving substitution of one or more amino acids. Efforts have also been made to study in detail the function of different regions of a protein. Following are some results of such efforts β-lactamase. This enzyme functions in the periplasmic space of bacterial cells. The enzymes hydrolyses and inactivates the β-lactam ring of penicillin derivatives and help in transport across the inner membrane. During transport a polypeptide (signal sequence peptide of 23 amino acids) is cleaved off. Detailed analysis suggested that transport and processing does not depend on this polypeptide of 23 amino acids alone. An active site involving amino acid serine has also been identified, since its replacement by cysteine leads to reduction in the activity of this enzyme.
Dihydrofolate reductase. In this enzyme, replacement of a single amino acid, aspartic acid (ASP) by asparagine (ASN), led to a decrease in specific acitivity by a thousand fold, suggesting that aspartic acid is very important for the active site. Other similar modifications were also examined. Insulin. It consists of A and B chains linked by C-peptide of 35 amino acids. It was shown that a sequence of 6 amino acids for C-peptide was adequate for the linking function Lactose Permease (product of y gene of lac operon). This enzyme is involved in transport of lactose and a cysteine to glycine substitution showed that this amino acid was not essential for transport. Further, out of four histidine residues, two at positions, 35 and 39 do not play any essential role in transport, while the mutation in any of the other two histidines at positious 208 and 322, lead to loss of transport function.
T4 lysozyme. A mutation of isoleucine to cysteine in this enzyme leading to formation of a disulphide bridge led to thermal stability and a 200 fold increase in enzyme activity even at 67°C Human β interferon. Removal of one of the three cysteine residues led to an improvement in stability of the enzyme λ repressor. The protein could be engineered to develop a specific site for cro-protein, since the alteration led to development of a cro recognition site Acetylcholine receptor. This protein is involved in transport of acetylcholine through the membrane. Specific regions of this protein involved in acetylcholine binding and channel formation have been identified Cytochrome C. A phenylalanine residue has been identified to be non-essential for electron transfer but is involved in determining the reduction potential of the protein Trypsin. It could be redesigned to have altered substrate specificity.	Your Ad Here
Subtilisin. Another successful alteration of substrate specificity involved the enzyme subtilisin reported in 1986-87 Lactate dehydrogenase. A lactate dehydrogenase (LDH) from Bacillus stearothermophilus was modified separately by each of the three substitutions of amino acids (resulting from mutation; Asp¹⁹⁷® Asn; Thr²⁴⁶® Gly; Gln¹⁰²®Arg.) The Substitution, Gln¹⁰²®Arg. led to changes in specificity from lactate to malate, high efficiency, comparable to that which the native LDH had for lactate Lactic protease. Substrate specificity of lactic protease (in E. coli), has been shown to be dramatically modified by replacing active site methionine by alanine (Met192® Ala; Met213® Ala)

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Methods for Protein Engineering Site Directed Mutagenesis in Vivo Gene Modification Through Oligonucleotide Synthesis Breeding a Better Catalyst Using Random Events Artificial Random Mutagenesis Random DNA Shuffing Using Recombination Combination of Structure Based and Envolutionary Approaches Multienzyme Systems bi and Polyfunctional Enzymes by Gene Fusion Chemical Modification of Enzymes Production of Site Specific Nucleases Production of Artifical Semi Synthetic Oxido Reductases Flavo Enzyme Hybrid Enzymes Combinatorial Chemistry Organic Synthesis Libraries De Novo Design of Catalysts Some Early Achievements of Protein Engineering Possible Candidates for Future Protein Engineering Improving Enzymes by Using Organic Solvents

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