Protein Engineering

By modifying the proteins gene sequence, researchers can change protein structure to enhance protein function in specific ways. Change can include: Increased stability such as: o Resistance to degradation o pH change o temperature o oxidation o contamination Enhanced enzyme activity A change in substrate specificity or enzyme activity in extreme or less-than-optimum conditions Increased nutritional value Several methods are available for changing the amino acids of proteins by mutating the corresponding nucleotides of the DNA. One method, oligonucleotide-directed mutagenesis, uses a short, single-strand DNA, an oligonucleotide 15 to 20 bases long, that is complementary to the gene region to be mutated except for the individual bases to be changed. The mutagenesis process is outlined in the following: 1. The gene to be mutated must first be cloned. A viral vector, the bacteriophage M13, is used in the cloning process. M13 has a both single-strand DNA stage that is packaged within a viral coat and a double-strand phase, or replicative form (RF) that is synthesized after cell infection. The RF, which is structurally similar to a plasmid, is used for cloning the DNA to be mutated. From these recombinant molecules, single-strand molecules are then generated for the mutagenesis process. 2. The oligonucleotide containing a specific change in sequence is then annealed to the singlestrand circular DNA molecule, except at the specific mismatch site that cannot base pair with it. 3. The oligonucleotide serves as a primer for replication by DNA polymerase, and DNA ligase seals the ends of the newly synthesized circular DNA. The resulting molecule is a double-strand, circular DNA with a mismatch region where the oligonucleotide mutation is located. 4. The DNA is transferred to E. coli, where it is replicated. One strand of the original DNA molecule has the mutation from the oligonucleotide and the other does not. Thus, some replicated RF double-strand molecules will have the mutation in their DNA sequence and the others will not; the latter are called wild type. 5. Progeny phage are produced within the bacterial host cells and are released as a single-strand DNA packaged in a viral protein coat. 6. Mutant plaques are determined by hybridizing the oligonucleotide (used to generate the mutation) to the resulting plaques containing either wild-type or mutant DNA. Because this mutagenesis procedure is inefficient and generates far fewer mutant molecules than expected, methods for enriching mutated DNA are used. Once obtained, the double-strand mutant

DNA (gene or cDNA) is isolated, placed in an expression vector, transferred to appropriate host cells such as bacteria or yeast, and expressed as a mutant protein for further study. Another way t mutate proteins is to substitute a DNA fragment, or cassette, containing the selected nucleotide mutations for the same DNA fragment in the organism. The DNA region containing the nucleotides to be changed is removed with a restriction enzyme and replaced with the cassette. This replacement is conducted in vitro in a vector that is then transferred to E. Coli for replication. A variety of other mutagenesis methods are used, some technically complex and using PCR, such as PCR-amplified oligonucleotide directed mutagenesis. The method used depends on the researchers goal: to obtain random, multiple-point mutations by producing single nucleotide changes at different intervals in the DNA; to introduce deletions or insertions of nucleotides; or, if the exact nucleotides to be changed are unknown, to generate a series of DNAs with different mutations in a specific area for further study.

Alecxandra O. Ventura III-Mendel