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Protein engineering can be defined as a collection of recombinant DNA techniques (including but not exclusively gene mutagenesis) that result in directed alterations being made to protein molecules, often to improve the properties of enzymes used in industrial processes. • Engineered proteins are sometimes referred to as factitious proteins. Prerequisite steps in protein engineering The successful engineering of proteins requires an understanding of the basic concepts of proteins from expression to composition.
1. The protein engineer is required to begin by finding the protein of interest hidden
within a mixture of proteins.
2. After locating the desired protein, the protein engineer must be able to clone and
express it for further analysis.
3. Before engineering the protein of interest, the characteristics of the wild-type protein
must be determined from a variety of analytical methods. 4. Mutant proteins are then produced to assess elements of the protein that are necessary for function.
5. With the functional sites identified, the protein engineer begins to evaluate
alterations in these sites that result in the desired new properties. properties may include Alterations in stability Alterations in catalytic activity Alterations in receptor binding Alterations in specificity Alterations in pharmacokinetic properties Alterations in immunogenicity These new
6. The protein engineer must have a clear understanding of the required improvements
and their impact on the intended use of the protein. Necessities of protein engineering The very underpinnings of the biotechnology industry with regard to proteins are founded upon protein engineering research.
The applications of protein engineering range from creating structural motifs such as alpha-helical bundles or leucine zippers that test theories of protein folding and protein stability, to the production of first- and second-generation protein products for human therapeutics or industrial products. These products include injectable proteins such as insulin, growth hormone, erythropoietin, hematopoietic cytokine granulocyte-colony stimulating factor, and viral subunits of hepatitis B, as well as
enzymes for improved food production, biosensors, pollution control, and even biocomputing.
The de novo design of new proteins with prescribed properties will eventually result in even more useful products. Several fundamental questions currently being addressed by protein engineers involve - Catalysis - Molecular recognition (e.g. substrate specificity and protein-ligand interactions) - Protein folding - Protein stability - Protein-protein interactions.
In studying these questions, some classic approaches are utilized by protein engineers to target those amino acids that potentially play functional or structural roles and then delete or replace them with alternate residues to test the role of steric constraints, hydrophobic forces, electrostatics and charge, and the placement of hydrogen bonds, salt bridges, disulfide bonds, water, or metals.
A database of information for a particular protein or class or proteins can be established in this fashion. In certain cases, a database may give a researcher clear clues regarding how to pro duce a protein with a predictable change in structure or function.
Fields of protein engineering The five broad categories of protein engineering projects are as follows: 1. Protein structure and function a) Signal sequences Targeting to organelles Secretion Substrate binding Transition state stabilization Allostery and cooperativity Mechanism Folding pathway Energetic contributions Subunit enteractions Increased thermal and solvent stability
b) Enzyme properties
c) Polypeptide folding and stability
2. Construction of industrially useful proteins
Alteration of substrate specificity Novel enzyme reactions Spectroscopy (ESR, NMR, fluorescence) X-ray crystallography
3. Introduction of reporter groups
4. Design of novel drug carriers
5. Design and production of clinically useful proteins, such as insulin, cholesterol esterase,
interferon, etc. Basic methods of protein engineering 2002(5.0)
1. 2. 3.
Genes or cDNAs encoding proteins of interest can be cloned from natural sources or synthesized de novo from the known protein sequences. The DNA sequences can then be genetically engineered to encode redesigned protein with insertions, deletions or precisely placed amino acid substitutions. These altered genes can then be introduced into appropriate heterologous expression systems to overproduce the engineered proteins to reagent-level quantities and qualities.
The proteins can be purified and analyzed both biochemically and biophysically to determine the effect of the amino; acid modification.
Finding protein of interest 1. The access to the wild type protein of interest is typically in limited quantities.
If the wild type protein is not available, but its sequence has been determined, cDNA can be synthesized, cloned into the appropriate plasmid, and expressed in the desired host.
3. In the case of discovery research, the protein of interest may not be well defined.
If a homologous sequence (e.g.
same family or different species) is known, cDNA
probes from these molecules are often used to locate the oligonucleotide sequence encoding the desired protein within a cell that is producing the protein.
For discovery research, homologous proteins may not be known, but a ligand or cofactor may be available. The ligand is then used to locate the protein of interest through radio-labeling and immunoprecipitation methods.
Once sufficient quantities (~ 100 µg to 1 mg) of the wild type protein are available, it can be used to generate monoclonal and polyclonal antibodies, utilized for subsequent assays and purification.
Developing recombinant DNA libraries
⇒ The creation of a DNA library that includes a full-length copy or overlapping proteins of
the genes/cDNA of interest is no longer the rate-limiting step in most cloning efforts.
⇒ DNA libraries are of two types; genomic libraries and cDNA libraries.
⇒ Useful genomic DNA or cDNA libraries are readily available from virtually any species or tissues.
⇒ Where a particular library is not available, a commercial source can be relied upon for
standardized library construction kits or even for customized construction of the entire library itself. Site directed Mutagenesis ⇒ Methods for making a precise alteration in a gene sequence in order to change the structure and possibly the activity of a protein
Changes in protein structure can be engineered by site-directed mutagenesis techniques, which result in defined alterations being made to the nucleotide sequence of the gene coding for a protein of interest. These techniques enable the functions of different parts of a protein to be examined, and also have widespread importance in the development of new enzymes for biotechnological purposes.
Conventional mutagenesis is a random process that introduces changes at unspecified positions in a DNA molecule.
Screening of large numbers of mutated organisms is necessary to find a mutation of interest. Even with microbes, which can be screened in huge numbers, the best that can be hoped for is a range of mutations in the correct gene, one of which might affect the part of the protein being studied. Site-directed mutagenesis offers a means of making much more specific mutations. The most important of these methods are
Oligonucleotide-directed mutagenesis Artificial gene systhesis PCR
Oligonucleotide-directed mutagenesis In this technique, an oligonucleotide containing the desired mutation is annealed to a single-stranded version of the relevant gene, the latter obtained by cloning in an M13 vector. The oligonucleotide primes a strand synthesis reaction that is allowed to continue all the way around the circular template molecule (Fig 1A).
After introduction into E. coli, DNA replication produces numerous copies of this recombinant DNA molecule, half of these being copies of the original strand of DNA, and half copies of the strand that contains the mutated sequence. All of these double-stranded molecules direct synthesis of phage particles, so about half the
phages released from the infected bacteria carry a single-stranded version of the mutated molecule. The phages are plated onto solid agar so that plaques are produced, and the mutant ones identified by hybridization probing with the original oligonucleotide (Fig 1B).
Figure 1: Oligonucleotide-directed mutagenesis
The mutated gene can then be placed back in its original host by homologous recombination, or transferred to an E. coli vector designed for synthesis of protein from cloned DNA, so that a sample of the mutated protein can be obtained.
Artificial gene synthesis involves constructing the gene in the test tube, placing mutations at all the desired positions. The gene is constructed by synthesizing a series of partially overlapping oligonucleotides, each up to 150 nucleotides in length. The gene is assembled by filling in the gaps between the overlaps with DNA polymerase, and ligated into a cloning vector prior to introduction into the host organism or into E. coli.
PCR can also be used to create mutations in cloned genes, though like oligonucleotide-directed mutagenesis, only one mutation can be created per experiment. The method shown in Figure 2 involves two PCRs, each with one normal primer (which forms a fully base-paired hybrid with the template DNA),
and one mutagenic
corresponding to the mutation).
Fig 2: One method for site-directed mutagenesis by PCR. This mutation is therefore initially present in two PCR products, each corresponding to one half of the starting DNA molecule. The two PCR products are then mixed together and a final PCR cycle carried out to construct the full-length, mutated DNA molecule.
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