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The protein manufacturing cost remains high and there is a growing demand to develop cost efficient and rapid protein purification methods. Understanding of the different protein purification methods and optimizing the downstream processing are critical to minimize production costs while maintaining the quality of acceptable standards of homogeneity. [2]
DNA-binding proteins are proteins that have DNA-binding domains and thus have a specific or general affinity for single- or double-stranded DNA. [ 3 ] [ 4 ] [ 5 ] Sequence-specific DNA-binding proteins generally interact with the major groove of B-DNA , because it exposes more functional groups that identify a base pair .
The DNA will form a pellet at the bottom of the solution, while the contaminants will remain in the liquid. Purification: After the DNA is precipitated, it is usually further purified by using column-based methods. For example, silica-based spin columns can be used to bind the DNA, while contaminants are washed away.
Another use for affinity chromatography is the purification of specific proteins using a gel matrix that is unique to a specific protein. For example, the purification of E. coli β-galactosidase is accomplished by affinity chromatography using p-aminobenyl-1-thio-β-D-galactopyranosyl agarose as the affinity matrix. p-aminobenyl-1-thio-β-D ...
The purity and amount of protein can be assessed by methods such SDS-PAGE and Western blotting. [14] [10] [15] Affinity purification using a polyhistidine-tag usually results in relatively pure protein. Protein purity can be improved by the addition of a low (20-40 mM) concentration of imidazole to the binding and/or
HUH-tag, a sequence-specific single-stranded DNA binding protein that covalently binds to its target sequence; Maltose binding protein-tag, a protein which binds to amylose agarose [26] Nus-tag; Thioredoxin-tag; Fc-tag, derived from immunoglobulin Fc domain, allow dimerization and solubilization. Can be used for purification on Protein-A Sepharose
DNA-binding domains with functions involving DNA structure have biological roles in DNA replication, repair, storage, and modification, such as methylation. [citation needed] Many proteins involved in the regulation of gene expression contain DNA-binding domains. For example, proteins that regulate transcription by binding DNA are called ...
Protein purification tags: Some expression vectors include proteins or peptide sequences that allows for easier purification of the expressed protein. Examples include polyhistidine-tag, glutathione-S-transferase, and maltose binding protein. Some of these tags may also allow for increased solubility of the target protein.
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