Protein Engineering

One of the most powerful developments in molecular biology has been the ability to artificially create defined mutations in a gene and analyse the resulting protein following in vitro expression. Numerous methods are now available for producing site-directed mutations, many of which now involve the PCR. Commonly termed protein engineering , this process involves a logical sequence of analytical and computational techniques centred around a design cycle. This includes the biochemical preparation and analysis of proteins, the subsequent identification of the gene encoding the protein and its modification. The production of the modified protein and its further biochemical analysis completes the concept of rational redesign to improve or probe a protein’s structure and function ( Figure 1).

Fig1. Protein design cycle used in the rational redesign of proteins and enzymes.

The use of design cycles and rational design systems are exemplified by the study and manipulation of subtilisin. This is a serine protease of broad specificity and of considerable industrial importance being used in soap powder and in the food and leather industries. Protein engineering has been used to alter the specificity, pH profile and stability to oxidative, thermal and alkaline inactivation. Analysis of homologous thermophiles and their resistance to oxidation has also been improved. Engineered subtilisins of improved bleach resistance and wash performance are now used in many brands of washing powders. Furthermore, mutagenesis has played an important role in the re-engineering of important therapeutic proteins such as the Herceptin ® antibody, which has been used to successfully treat certain types of breast cancer.

Kunkel Oligonucleotide-Directed Mutagenesis

This is a traditional method of site-directed mutagenesis and demands that the gene is already cloned. Complete sequencing of the gene is essential to identify a potential region for mutation. Once the precise base change has been identified, an oligonucleotide is designed that is complementary to part of the gene, but has one base difference. This difference is designed to alter a particular codon, which, following translation, gives rise to a different amino acid and hence may alter the properties of the protein.

A single-stranded uracil-containing vector such as M13 is grown in a selectable host deficient of the enzymes dUTPase ( dut^- ) and uracil N -deglycosidase ( ung^- ). The mutagenic oligonucleotide and the single-stranded vector are annealed and DNA polymerase is added together with the dNTPs. The primer for the reaction is the 3′ end of the oligonucleotide. The DNA polymerase produces a new DNA strand complementary to the existing one, but which incorporates the oligonucleotide with the base mutation. The subsequent transformation of a dut ⁺ ung ⁺ E.coli strain with the recombinant produces multiple copies, one strand containing the sequence with the mutation and the other the parent uracil-containing strand which is preferentially degraded.

Plaque hybridisation using the oligonucleotide as the probe is then used at a stringency that allows only those plaques containing a mutated sequence to be identified ( Figure 2). Further methods have also been developed that simplify the process of detecting the strands with the mutations.

Fig2. Oligonucleotide-directed mutagenesis. This technique requires a knowledge of nucleotide sequence, since an oligonucleotide may then be synthesised with the base mutation. Annealing of the oligonucleotide to complementary (except for the mutation) single-stranded DNA provides a primer for DNA polymerase to produce a new strand and thus incorporates the primer with the mutation.

PCR-Based Mutagenesis

The PCR has been adapted to allow mutagenesis to be undertaken and this relies on single bases mismatched between one of the PCR primers and the target DNA becoming incorporated into the amplified product following thermal cycling.

The basic PCR mutagenesis system involves the use of two primary PCR reactions to produce two overlapping DNA fragments, both bearing the same mutation in the overlap region; this technique is thus termed overlap extension PCR. The two separate PCR products are made single-stranded and the overlap in sequence allows the products from each reaction to hybridise. Subsequently, one of the two hybrids bearing a free 3′-hydroxyl group is extended to produce a new duplex fragment. The other hybrid with a 5′-hydroxyl group cannot act as substrate in the reaction. Thus, the overlapped and extended product will now contain the directed mutation (Figure3). Deletions and insertions may also be created with this method, although the requirements of four primers and three PCR reactions limits the general applicability of the technique. A modification of overlap extension PCR may also be used to con struct directed mutations; this is termed megaprimer PCR . This latter method utilises three oligonucleotide primers to perform two rounds of PCR. A complete PCR product, the megaprimer is made single-stranded and this is used as a large primer in a further PCR reaction with an additional primer.

Fig3. Construction of a synthetic DNA fragment with a predefined mutation using overlap PCR mutagenesis.

The above are all methods for creating rational defined mutations as part of a design cycle system. However, it is also possible to introduce random mutations into a gene and select for enhanced or new activities of the protein or enzyme it encodes. This accelerated form of artificial molecular evolution may be undertaken using error-prone PCR , where deliberate and random mutations are introduced by a low-fidelity PCR amplification reaction. The resulting amplified gene is then translated and its activity assayed. This has already provided novel evolved enzymes such as a p -nitrobenzyl esterase, which exhibits an unusual and surprising affinity for organic solvents. This accelerated evolutionary approach to protein engineering has been useful in the production of novel antibodies produced by phage display and in the development of antibodies with enzymatic activities ( catalytic antibodies).

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مواضيع ذات صلة

Expression of Foreign Genes

cDNA Libraries

Genomic DNA Libraries

Ligating DNA Molecules

Knockout of Leptin Gene

Engineered CRISPR–Cas9 Systems Allow Precise Genome Editing

Plasmid Expression Vectors Can Be Designed for Use in Animal Cells

Engineering Antibody Synthesis in Bacteria

Heritable Genome Editing

Challenges to in Vivo Editing of Hematopoietic Stem Cells

EX Vivo Manufacturing of Therapeutic Cells

Using Engineered Nucleases to Initiate the Genome Editing Process