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PubMed1991

New molecular biology methods for protein engineering.

This review outlines recent advances in the application of molecular biological techniques to the study of protein structure and function. The chapter is divided into four main sections: methods for oligonucleotide-directed mutagenesis; mutational strategies for identifying functional residues and domains; systems for expression; and future developments. Few new methods were reported in 1990; however, a number of the papers that appeared represent refinements of previously reported strategies. This review is also published in Current Opinion in Structural Biology 1991, 1:605-610.

Biotechnology↗
PubMed1992

Protein engineering of Drosophila alcohol dehydrogenase. The hydroxyl group of Tyr152 is involved in the active site of the enzyme.

Drosophila alcohol dehydrogenase is the most studied member of the family of short-chain alcohol dehydrogenases, although its tridimensional structure still remains unknown. We have engineered a Drosophila alcohol dehydrogenase in which tyrosine-152, an invariant residue in all members of the family, has been substituted by phenylalanine. The mutated gene has been expressed in yeast and pure mutant enzyme has been prepared by a one-step FPLC chromatographic procedure. Drosophila alcohol dehydrogenase-phenylalanine-152 shows no enzymatic activity. This result suggests not only that tyrosine-152 could constitute an essential building block of the active site but also that its hydroxyl group is directly involved in the redox reaction catalyzed by the enzyme.

Alcohol Dehydrogenase↗
PubMed1992

Protein engineering of the milk-clotting aspartic proteinases.

Calf Chymosin and a fungal protease from Mucor pusillus (Mucor rennin) are members of the aspartic proteinases used as milk-coagulants in cheese industry. A system for production of recombinant chymosin as inclusion bodies in Escherichia coli cells and its refolding into the active form was established. Another expression system for production of Mucor rennin in Saccharomyces cerevisiae was also established. Mucor rennin was efficiently excreted from the yeast host as a heavily glycosylated form. Glycosylation affected both the secretion and the enzyme properties. Site-directed mutagenesis of the Tyr residue at position 75 in chymosin and Mucor rennin revealed its crucial role in catalytic function of the aspartic proteinases. The results also suggested possibility to improve practical properties of the milk-clotting enzymes by site-directed mutagenesis.

Amino Acid Sequence↗
PubMed1992

Protein engineering of antibodies.

This article reviews the technical advances in antibody engineering and the clinical applications of these molecules. Recombinant DNA technology facilitates the construction and expression of engineered antibodies. These novel molecules are designed to meet specific applications. Although genomic and cDNA cloning have been used widely in the past to isolate the relevant antibody V domains, at present, the PCR-based cloning is the preferred system. Bacterial and mammalian expression systems are used commonly for the production of antibodies, antibody fragments, and antibody fusion proteins. A range of chimeric antibodies with murine V domains joined to C regions from human and other species have been produced and found to exhibit the expected binding characteristics and effector functions. Humanized antibodies have been developed to minimize the HAMA response, and bifunctional immunoglobulins are being used in tumor therapy and diagnosis. Single chain antibodies and fusion proteins with antibody specificities jointed to nonimmunoglobulin sequences provide a source of antibody-like molecules with novel properties. The potential applications of minimal recognition units and antigenized antibodies are described. Combinatorial libraries produced in bacteriophage present an alternative to hybridomas for the production of antibodies with the desired antigen binding specificities. Future developments in this field are discussed also.

Amino Acid Sequence↗
PubMed1992

Multidisciplinary cycles for protein engineering: site-directed mutagenesis and X-ray structural studies of aspartic proteinases.

The specificity and pH profile of aspartic proteinases have evolved to include not only pepsin with a broad specificity and an optimal activity in acid media, but also renin, with high specificity for angiotensinogen and activity close to neutral pH. Comparisons of the structures and catalytic activities of aspartic proteinases provide helpful clues for engineering new activity profiles. We illustrate an approach that involves recombinant DNA techniques, biochemistry, structure determination and biocomputing. We use the 3-D structures of inhibitor complexes of several aspartic proteinases to define likely intermediates and specificity sub-sites. The multidisciplinary research is organised as cycles, in which each cycle tests a design hypothesis proposed in the previous cycle. We use one member of the aspartic proteinase family, chymosin, to illustrate these ideas in engineering enzymes with altered pH optima and specificities.

Amino Acid Sequence↗
PubMed1992

Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus: functional and structural consequences of mutation at the S4 substrate binding pocket.

Serine endoproteases such as trypsins and subtilisins are known to have an extended substrate binding region that interacts with residues P6 to P3' of a substrate. In order to investigate the structural and functional effects of replacing residues at the S4 substrate binding pocket, the serine protease from the alkalophilic Bacillus strain PB92, which shows homology with the subtilisins, was mutated at positions 102 and 126-128. Substitution of Val102 by Trp results in a 12-fold increase in activity towards succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPFpNA). An X-ray structure analysis of the V102W mutant shows that the Trp side chain occupies a hydrophobic pocket at the surface of the molecule leaving a narrow crevice for the P4 residue of a substrate. Better binding of sAAPFpNA by the mutant compared with the wild type protein as indicated by the kinetic data might be due to the hydrophobic interaction of Ala P4 of the substrate with the introduced Trp102 side chain. The observed difference in binding of sAAPFpNA by protease PB92 and thermitase, both of which possess a Trp at position 102, is probably related to the amino acid substitutions at positions 105 and 126 (in the protease PB92 numbering). Kinetic data for the variants obtained by random mutation of residues Ser126, Pro127 and Ser128 reveal that the activity towards sAAPFpNA increases when a hydrophobic residue is introduced at position 126.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Alteration of enzyme specificity and catalysis by protein engineering.

New substrate specificities can be introduced into existing enzymes for the purpose of making them more suitable for the chemoenzymic synthesis of single compound drugs and other chiral compounds. The most productive route used in the past year has involved the utilization of the catalytic and substrate-binding properties from homologous enzymes found in nature, one example being the broadening of the substrate specificity of yeast alcohol dehydrogenase. Other highlights include the creation of thermostable dehydrogenases that will interconvert NADPH and NADH, and the design of mutant enzymes with improved catalytic rates compared with their wild-type counterparts.

Amino Acid Sequence↗
PubMed1991

Enhancing the thermostability of glucose isomerase by protein engineering.

We have engineered recombinant glucose isomerase (GI) from Actinoplanes missouriensis by site-directed mutagenesis to enhance its thermal stability in both the soluble and immobilized forms. Substitution of arginine for lysine at position 253, which lies at the dimer/dimer interface of the GI tetramer, produced the largest stabilization under model industrial conditions. We discuss our results in terms of a model in which chemical glycation of lysines by sugars in the industrial corn syrup substrate represents a major pathway of destabilization.

Aldose-Ketose Isomerases↗
PubMed1992

Potential of genetic algorithms in protein folding and protein engineering simulations.

Genetic algorithms are very efficient search mechanisms which mutate, recombine and select amongst tentative solutions to a problem until a near optimal one is achieved. We introduce them as a new tool to study proteins. The identification and motivation for different fitness functions is discussed. The evolution of the zinc finger sequence motif from a random start is modelled. User specified changes of the lambda repressor structure were simulated and critical sites and exchanges for mutagenesis identified. Vast conformational spaces are efficiently searched as illustrated by the ab initio folding of a model protein of a four beta strand bundle. The genetic algorithm simulation which mimicked important folding constraints as overall hydrophobic packaging and a propensity of the betaphilic residues for trans positions achieved a unique fold. Cooperativity in the beta strand regions and a length of 3-5 for the interconnecting loops was critical. Specific interaction sites were considerably less effective in driving the fold.

Algorithms↗