Protein engineering of laundry detergent
The enzyme subtilisin is a protease (a protein-digesting protein) produced by bacteria, and has a broad specificity for proteins that commonly soil clothing. To improve the efficiency of laundry detergents, detergent manufacturers added subtilisin (and advertised it on the detergent box as "with biologically active enzymes" or with similar uninformative slogans). However, subtilisin is inactivated by bleach, and it was found that this inactivation was due to oxidation of the amino acid methionine at position 22 of the subtilisin molecule. Using site-directed mutagenesis of the subtilisin gene in E. coli, this methionine was changed to a variety of other amino acids, and the subtilisin activity and bleach resistance of the mutated protein was tested. It was found that substitution of methionine by alanine was the best in terms of activity and stability, and now many laundry detergents contain cloned, genetically engineered subtilisin. Something to remember next time you do your laundry....
Combinatorial mutagenesis
In biotechnology, one is often interested in creating enzymes with new specificities. For example, an enzyme that can recognize a different substrate that can be converted into a valuable product would be attractive from a biotechnological point of view. Generally, simple mutations (to be introduced by site-directed mutagenesis, for example) are not expected to have as drastic effects as altering an enzyme's substrate recognition pattern, as many amino acid residues in the enzyme (often not close to one another in the primary structure of the protein) affect the binding pocket of the substrate in the enzyme. Obviously, several amino acid residues may need to be altered simultaneously to achieve the goal of altering substrate specificities. However, as any amino acid residue may be altered into 19 other ones, the number of amino acid combinations that can be made if mutations are introduced at various residues simultaneously can become very large. For example, if four amino acid residues are altered simultaneously, there are 19-to-the-power-of-4 (that is, over 100,000) different combinations in which this can occur.
Generally, it is unknown which of these combinations is what one is looking for, as it is difficult to predict on the basis of a primary sequence what the three-dimensional structure of a protein (and of the active site) will be in detail. Therefore, instead of humans trying to decide what might work best, the best progress is often made by having essentially all different combinations made at the DNA level in different plasmids (can be done using degenerate oligonucleotides), use all these different plasmids as a mixture to transform E. coli, have E. coli express the different proteins (each E. coli cell and its clones will express one particular protein assuming it has taken up one plasmid molecule), and select the E. coli cell(s) that may be able to convert a new substrate. The latter part of the process often is the most difficult, as one does not really want to test a zillion of different E. coli clones on whether or not they can convert a particular substrate. Instead, one may choose selection methods, depending on the properties of the protein that has been altered. For example, if the protein is likely to be on the outside of the E. coli cell, one can select clones with proteins with high affinity for the new substrate by attaching the new substrate covalently to a column, wash E. coli over the column, and cells that come off slowest are likely to have protein with affinity for the substrate.
Combinatorial mutagenesis does not limit itself to applications involving DNA. Peptides can also be synthesized from a degenerate mix of amino acid analogs, and the resulting mix of peptides can be screened for desired properties, in particular pharmaceutical applications. Moreover, RNA can be synthesized combinatorially, and degenerate RNA mixtures have been used to study features that are needed to provide RNA with catalytic properties. In any case, combinatorial mutagenesis provides virtually limitless possibilities for genetic engineering, and has become an important tool in biotechnology.
http://photoscience.la.asu.edu/photosyn/courses/BIO_343/
This web site contains the syllabus for the MBB 343/BIO 343 course, describes the laboratory experiments, and covers materials that will be discussed in the lecture but that have not been covered sufficiently in the required textbook (B.R. Glick and J.J. Pasternak (1998) Molecular Biotechnology: Principles and Applications of Recombinant DNA, second edition).
Most of the material at this website has been compiled over the years by Wim Vermaas, a Professor in the Plant Biology Department. Instructors this year are Wim Vermaas (weeks 5-8, 12-13, and 15-16) and David Rhoads (weeks 1-4, 9-11, and 14).
Contact information:
Wim Vermaas: office location: LS-E549; phone: (480) 965-3698; Email: wim@asu.edu; office hours for Fall 2002: M and TH 10:40-11:30.
David Rhoads: office location:LS-E511; phone (480) 965-2583; Email: david.rhoads@asu.edu; office hours for Fall 2002: T/TH 1:30-2:30 pm on the days that he lectures only.
Material at this site may be used freely by others as long as the source of the information is acknowledged. Contact the appropriate web master to learn about the rules of using materials contained at other web sites that are referred to at this site.