9. PROTEIN ENGINEERING AND
SITE-DIRECTED MUTAGENESIS (SDM)

A mutation is any change in the base sequence of DNA - deletion, insertion, inversion, substitution. A mutant is a cell or organism carrying a mutation. Mutagenesis is the process of producing mutations. Mutations can occur spontaneously, although the rate of mutation can be increased by various factors and agents (called mutagens) but the results are entirely random and not reproducible.

Mutagens include:

• ionizing radiations (e.g. X-rays, gamma rays)

• non-ionizing radiations (e.g. ultraviolet light - which is not strictly speaking a mutagen - see explanation)

• various chemicals (e.g. mustard gas, benzene, ethidium bromide)

The above have been used in the past to deliberately produce random mutations in the DNA of organisms in attempts to improve them - a process known as strain improvement. For example, the mould that produces penicillin, Penicillium chrysogenum, has been assaulted since the 1940s by every conceivable type of mutagen, but especially UV light and certain chemicals. The mutants have then been screened and selected for improvements such as increased yield. This is quite a success story since the yield of penicillin from industrial strains of the organism is millions of times higher when compared to the yield from the original wild-type mould isolated from a mouldy melon bought in a market.
Q. What type of melon was it?

Strain improvement has also been practiced for centuries by man with farm and domestic animals and crop plants (Think: a St Bernard dog and a Pekinese are the same species and have the same ancestor in the wolf!). Up to now, though, man has had to rely on spontaneous mutations and artificial selection to produce changes in a breed. The main problem with this approach is that it is very slow, particularly with higher organisms with long life cycles, and the desired result is never guaranteed (very 'hit-and-miss') because mutations are random and most are disadvantageous to the organism. Nowadays GM and Recombinant DNA Technology offer ways of speeding up the process and making it far more specific:

In vitro mutagenesis is mutation of DNA outside of a host cell. Site-directed mutagenesis (SDM), sometimes called site-specific mutagenesis, is a process that produces mutations in DNA that are controlled by us. In other words, we can decide what mutations we want and then produce them. This means that we can change, not only the base sequence of DNA and, hence, the genetic code of a gene, but also the gene product (the protein). We can, for instance, decide to change a particular amino acid in a polypeptide chain of a protein/enzyme by changing the codon in the gene coding for that amino acid. Changing the amino acid will often change the properties of the protein/enzyme (but not always for the better). This is one of the techniques that can be used in protein engineering - one of the most sophisticated applications of recombinant DNA technology - where the properties of a protein, such as an enzyme, are altered in an attempt to 'improve' it by changing (mutating) the gene coding for the protein using SDM. Desired improvements might be increased thermostability, altered substrate range, reduction in negative feedback inhibition, altered pH range, etc.

However, before we look at SDM, let us consider that proteins and enzymes can also be improved by chemically or physically treating a gene product (a protein) to alter it rather than altering the gene itself. Read this document.

1. Isolate the gene coding for the protein/enzyme, e.g. via mRNA and its conversion into cDNA.

2. Sequence the gene.

3. Decide on mutation that will "improve" the enzyme. This is usually based on study of the enzyme’s structure (1^o, 2 ^o, 3 ^o, i.e. 3-dimensional structure and amino acid sequence).

This is predictive and computer modelling is often used.

Usually one codon is altered at a time, i.e. only 1, 2 or, at the most, 3 bases are altered.

4. Use site-directed mutagenesis (SDM) to produce the desired change in codon(s) in the gene and subsequently the amino acid sequence of the enzyme molecule.

5. Test "new" enzyme for "improvement".

6. Open the champagne or weep into handkerchief!

There are now several methods for carrying out SDM, including PCR-based ones. The following one is called the single-primer method and is based on the use of the phage vector M13.

1. Isolate required enzyme gene, e.g. via mRNA and its conversion into cDNA.

2. Sequence the DNA of the gene (in order to decide on change required for primer in stage 5).

3. Splice gene into M13 vector dsDNA and transduce E. coli host cells.

4. Isolate ssDNA in phage particles released from host cells.

5. Synthesize an oligonucleotide primer with the same sequence as part of the gene but with altered codon (mismatch/mispair) at desired point(s). For example, one of the codons in DNA coding for the amino acid Alanine is CGG. If the middle base is changed by SDM from G to C the codon sequence becomes CCG which codes for a different amino acid (Glycine).

6. Mix oligonucleotide with recombinant vector ssDNA.

Carried out at low temperature (0-10^oC) and in high salt concentration to allow hybridization between oligonucleotide and part of gene. Under these low stringency conditions hybridization occurs despite the mismatch. Remember that complementarity between two pieces of ssDNA does not have to be 100% for hybridization to occur if stringency is low.

7. Use DNA polymerase to synthesize remainder of strand. (Oligonucleotide acts as a primer for the DNA synthesis.) Then add ligase  to make join between primer and new strand permanent ....

  dsDNA molecule. 

8. Transform E. coli cells and allow them to replicate recombinant vector molecule.

9. DNA replication is semi-conservative, therefore two types of clone are produced each of which excretes phage particles containing ssDNA:

Type 1: contain the wild-type gene (i.e. unaltered)

Type 2: contain the mutated gene!!!

DIAGRAM OF SDM USING
M13 PHAGE VECTOR

Ratio of the two types should be 1:1 but is not usually because E. coli "edits out" some of the mismatches.

10. Select mismatch clones bearing the mutation (How???).

11. If gene expression is required, use these clones to extract mutated DNA and insert it into an expression vector system with appropriate promoter, etc. to produce the modified gene product ('designer protein').

Useful web sites with further information about protein engineering:

Computational protein engineering

Hemi-humanized single-chain Fv against the CD18 surface antigen

END OF SECTION 9.
NOW GO TO SECTION 10
(DNA SEQUENCING).

Why UV light isn't a mutagen!

Contrary to popular belief, UV light does not directly cause mutation but does indirectly because it damages DNA. In some cells or species this is then repaired by special enzymes which excise the damaged DNA and then repair it - sometimes making mistakes in the process by incorporating the wrong base - hence a mutation is produced.

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Cantaloupe!!!

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Use the same oligonucleotide (which originally served as a primer in Stages 5 and 6) as a probe, i.e. add a label to it. The probe must now be used under conditions of high stringency (rather than low stringency as it was when used as a primer) to ensure that it only hybridizes with the altered DNA sequence of the desired clones and not with the DNA of clones containing the wild-type gene.

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