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The experimental stages are described as follows: Cloning: Cloning I and Cloning II. Protein Expression and Solubility Studies: Expression and Solubility I, Protein Purification: Purification and Solubility II, Protein Crystallography: In Crystal Trials, Crystallized, Diffracting Crystal, Data Collected, Phased, Refined and Deposited in PDB. Below they are described in general detail.
Cloning:
The first stage towards a protein structure is to take the desired gene of interest and clone it into an E. coli expression vector with a cleavable N- or C-terminal protein tag (eg. histidine6-Tag (His-Tag) or maltose-binding protein) for purification. Specific primers are designed to amplify the gene of interest by polymerase chain reaction (PCR), and then the PCR product is placed into a blunt cloning vector. The vector is sequenced to ensure that the Mtb gene sequence is correct. This gene is then extracted and ligated into various expression vectors, as mentioned above.
Protein Expression and Solubility Studies:
Protein expression is done by transforming the expression vector into various cell-lines (e.g. BL21 (DE3), BL21 (DE3) codon-enhanced and Rossetta cell-lines). Small-scale expressions are performed by optimizing E. coli host growth conditions and induction times. Expression levels are determined by analysis of whole cell lysates by SDS-page and Western blots.
Protein solubility studies are carried out to determine the most optimal buffer for protein solubility.
Protein Purification:
Once solublility conditions have been established, the construct is grown on a large-scale (4-L to 10-L) for protein purification. Affinity chromatography, gel-filtration, anion/cation-ion exchange chromatography are some of the methods used. Protein concentration is determined by several methods, Bradford, SDS-page, or OD at 280 nm. The precise molecular weight of all proteins is determined by mass spectrometry (MS), and to ensure the protein is folded the secondary structure of the protein is determined by circular dichroism (CD).
Fusion Protein Cleavage
If the protein tag has a tag, then it is usually removed if the protein is thought to have a higher oligomeric state than monomeric.
Protein Crystallography:
Once a pure soluble protein is obtained, spare matrix screens are setup using with hanging or sitting drop techniques. When crystalline material is observed, the condition is optimized to obtain single, well-diffracting crystals. Data on the crystal is usually collected in a synchrotron beam line after initial tests of diffractibility in the home laboratory. The structure maybe solved by molecular replacement if the protein of interest has a homologous (greater than 40% sequence identity) structure. If the protein structure is predicted to be novel, then heavy atom phasing is required. This requires either a selenomethionine-derivative of the protein, soaking protein crystals with heavy metals or a quick soak of protein crystals with sodium iodide etc. This is essential for solving the phases. The phase solution allows the construction of the electron density map. Once the electron density map is obtained, the protein model is built and refined and the coordinates deposited to the PDB.
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