Key Points- Expression, Cloning & Purification of Proteins PDF

**Key Points- Expression, Cloning & Purification of Proteins** **Expression** refers to whether or not a protein is produced in a cell or tissue. Constitutively expressed proteins are always present, whereas inducible proteins are only produced (expressed) under certain conditions, i.e their produc...

**Key Points- Expression, Cloning & Purification of Proteins** **Expression** refers to whether or not a protein is produced in a cell or tissue. Constitutively expressed proteins are always present, whereas inducible proteins are only produced (expressed) under certain conditions, i.e their production can be turned on and off. The best studied "on/off" switch is the ***lac operon***, a region of DNA in the *E.coli* bacterium which controls the production of the proteins associated with the use of lactose. These proteins are a *β-galactosidase* enzyme which hydrolyses lactose and a *permease* enzyme which allows lactose to pass through the bacterial membrane. A regulatory gene in the lac operon DNA produces a *repressor protein* which binds to the *operator* DNA region and switches off the production of the enzymes, since the RNA polymerase cannot travel from its binding region on the *promoter* region past the *operator* region to transcribe the genes which code for the enzyme. Lactose in the form of its isomer allo-lactose switches on the production of the enzymes by binding to the repressor protein and releasing it from the operator. IPTG is an allo-lactose mimic often used in laboratory studies of the lac operon. The *lac operon* is often used in the laboratory in **recombinant DNA** expression systems (**cloning vectors**) such as **plasmids** to act as an on/off switch to control the production of **recombinant proteins** following insertion of the foreign gene coding for the protein into the plasmid (done using restriction endonucleases and ligase enzymes). The plasmids also typically contain an antibiotic selection marker and a purification tag (e.g a His tag) at the N or C terminal to aid in purification of the recombinant protein. **Site directed mutagenesis** can be used with plasmids in the laboratory to change (mutate) specific amino acids in a recombinant protein. The Smith method uses a single stranded synthetic oligonucleotide (short piece of DNA) that is complementary to the foreign gene (DNA) in the plasmid for the recombinant protein, but with a base mutation in the sequence. The single stranded oligonucleotide containing the base mutation binds to the single stranded plasmid containing the foreign gene by base pairing and then is copied into a full double stranded plasmid by DNA polymerase. Replication of the plasmid then produces many plasmid copies with the mutated DNA sequence. An example of the use of site directed mutation is changing the Green Fluorescent Protein to a blue fluorescent protein. Another example is changing a yellow kiwifruit protease with low enzyme activity into a more active version similar to green kiwifruit protease, using the site directed mutation **P**75**Q.** The **P**75**Q** abbreviation means that a **P**roline amino acid in the original protein/enzyme at position 75 has been converted into the amino acid glutamine (**Q**). Proteins can be made in the laboratory *using **cell free expression systems*** which are cell extracts (e.g. from *E.coli* bacteria) made by breaking cells open, then centrifuging to obtain the supernatant. These supernatant extracts contain most of the components needed to make proteins (e.g. enzymes, ribosomes etc). When DNA (plasmid) and amino acids are added such systems allow rapid protein production. Site directed mutagenesis can also be used with cell free expression systems to incorporate unnatural amino acids into proteins (by converting a normal codon in the gene on a plasmid to a STOP codon (TAG), and then using a semi-synthetic t-RNA with the un-natural amino acid attached to bind to the stop codon on mRNA (UAG)): Examples of the use of unnatural amino acids include the use of O-2-bromoethyl-tyrosine as a "staple" to form an un-natural bond with cysteine to stabilise proteins (e.g. myoglobin) making them more resistant to heat denaturation and the use of 4-trifluoromethyl phenylalanine as an unnatural amino acid used in the 19F NMR study of proteins such as virus proteases. **A Reporter gene** is a section of DNA which researchers can attach to another gene of interest and which will then be expressed as a visual signal within a cell or tissue. Green fluorescent protein (GFP) DNA , originally from a jellyfish is often used for this purpose as is the DNA coding for the bioluminescent enzyme luciferase. **FRET** (Fluorescence Resonance Energy Transfer) is an energy transfer process which can occur between fluorescent proteins which are within 10nm of each other. The donor protein absorbs the excitation wavelength, and the acceptor protein then emits the emission wavelength. FRET can be used to study Protein-Protein Interactions (PPI) and enzyme activity. Other methods for studying PPI include Fluorescence Two Hybrid (F2H) and Yeast Two Hybrid (Y2H) assays. **Bioluminescence** can be produced when the enzyme luciferase reacts with its substrate luciferin in the presence of ATP. Bioluminescence is a technique that can be used in the laboratory to monitor reporter genes (luciferase) or to assay ATP using a luminometer (e.g. ATP in bacteria or blood cells -platelets). **Isolation and Purification of Proteins** Isolation of proteins from natural sources such as animal & plant tissues usually starts with disruption (breaking up) of the tissue using a blender (homogenisation). For organisms with tough cell walls (such as yeast) the sample is often ground with sand or frozen and ground with liquid nitrogen, or ultrasonic (sound waves) can be used. Shearing using a Potter-Elvehjem pestle can also be used with bacteria. During extraction the sample is kept cool and protease inhibitors may be added to prevent denaturation and protease enzyme activity which will degrade the proteins. Following tissue or cell disruption centrifugation is often used to separate the proteins in the aqueous extract from the tissue and cell debris. Preliminary separation of proteins from other molecules (sugars, lipids etc) in the initial extract is often done by "salting out" the proteins using ammonium sulfate or by size exclusion (Sephadex 25). These techniques can also be used to fractionate the proteins further. Once a crude protein extract is isolated, some type of **chromatography** is generally used to separate the different proteins in the extract. This can be based on protein charge (ion exchange chromatography), protein size (size exclusion chromatography), protein hydrophobicity (hydrophobic interaction chromatography-HIC) or affinity of the protein for a ligand (affinity chromatography). Often several of these techniques are used on the same sample. e.g. mussel carbonic anhydrase is purified by homogenisation & centrifugation of the mussel tissue, then salting out of the protein from the supernatant, followed by ion exchange chromatography and affinity chromatography of the proteins to obtain the purified carbonic anhydrase enzyme. Purification of recombinant (cloned) proteins is often more efficient than purification from natural sources as the proteins are present in higher concentrations and purification tags are added to aid purification during the cloning stage. The **His tag** consists of 6 histidine amino acids added to the recombinant protein to create a fusion protein. These His residues in the fusion protein can bind to a nickel containing column to separate the protein chromatographically. Once a sample has gone through the purification process it can be checked for purity using **electrophoresis** (SDS-PAGE for protein size, IEF for protein isoelectric point, or both SDS-PAGE & IEF combined in two dimensional electrophoresis). Further checks following SDS-PAGE can be made for the presence of the correct protein using specific antibodies for the protein in the Western blotting technique. Proteins can also be quantified using specific antibodies with enzymes linked to the antibody (ELISA). These enzyme linked antibodies produce a coloured or fluorescent product for quantification e.g. sandwich immunoassays. Protein scaffolds and nucleic acid aptamers are being developed as antibody alternatives which will work similarly to antibodies but do not require the use of animals in their production.