Studies on the improvement of functions of useful proteins

The advent of gene technology in the 1970s has led to the flourishing of the biotechnology industry. In the 1980s, attention was focused on biotechnology as a consequence of the enormous potential of recombinant DNA technology and genetic engineering. The expansion in commercial biotechnological activity has been based largely on a capacity to produce proteins in heterologous cell systems on an industrial scale. Protein engineering is a more recent development which is likely to have tremendous theoretical and practical implications. It can be used both to explore fundamental questions about protein folding, structure, and function, and to design useful proteins for medical and industrial applications. A flow chart of protein engineering is as follows: (1) cloning of the gene of an enzyme, (2) determination of the nucleotide sequence and/or amino acid sequence of the enzyme, (3) biochemical characterization of the enzyme, (4) determination of the three-dimensional structure of the enzyme, (5) molecular design of the enzyme, (6) alteration of the gene of the enzyme by site-directed mutagenesis, (7) characterization of the mutant enzyme, and then back to step (5). Since protein engineering is an interdisciplinary research field, cooperation among biologists, chemists, and physicists is essential for its success. In the near future, it will almost certainly become possible to alter the specific activity, substrate specificity, and optimum pH of enzymes, increase the thermal stability of proteins, and design proteins that can be used in nonaqueous solvents. In addition to the modification of existing proteins, the possibilities of designing entirely novel peptides and proteins with useful properties seem endless, since the amino acid sequences found in existing proteins represent only an infinitesimal fraction of all possible amino acid sequences. In this review, the productivity of homologous and heterologous gene products is discussed in detail. Chapter I shows an example of overproduction and the engineering of a diagnostic enzyme, cholesterol oxidase. Chapter II demonstrates an example of the genetic design of chimeric Escherichia coli as an overproducer of a mammalian protein, metallothionein. Chapter III deals with an example of an extracellular enzyme, pullulanase, analysis of structure-function relationships, and the engineering of starch-processing enzymes by recombinant DNA technology. In chapter IV, another example of overproduction and of a membrane-bound protein, monoamine oxidase, are shown. Finally, in chapter V an example of overproduction and the engineering of a cephem antibiotics-processing enzyme, GL7ACA acylase, are described. The aim of this investigation is to put into a real-world context the recombinant microorganisms used in the area of human therapeutics, the conventional food industry, and industrial processes. Current problems, how to overcome the limitations associated with the practical use of recombinant microorganisms, and strategies for creating a supermicrobe are discussed.