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Home >>Protein Engineering, Immunotoxins and Drug Designing - Engineering of Macromolecules >> Acheivements of Protein Engineering
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Acheivements 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 enzyme hydrolyses and inactivates the beta- lactam ring of penicillin derivatives and helps 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 add (ASP) by asparagine (ASN), led to a decrease in specific activity by a thousand fold, suggesting that aspartic acid is very important.(or 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, 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 positious 35 and '39 do' not play any essential role in transport, while the mutation in any of the other two histidines at positions 208 and 322, lead to loss of transport function.

T4 lysozyme. A mutation of isoleucine to cystine in this enzyme leading to formation of a disulphide bridge led to thermal stability and a 200 fold increase in enzyme activity even at 6T'C.

Human beta interferon. Removal of one of the three cysteine residues' I led to an improvement in stability of the enzyme.

λ repressor. This protein could be engineered to develop a specific site for cro protein, since the alteration led to development of a cro recognition site.I

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.

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 substitutiens of amino acids (resulting from mutations; Asp197... Asn; Thr246"'Gly; Gln102...Arg). The substitution, Gln102"'Arg, led to change in specificity from lactate to malate, with 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; Mer13-->Ala).
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