TAILIEUCHUNG - Protein Purification Part 12

Tham khảo tài liệu 'protein purification part 12', khoa học tự nhiên, công nghệ sinh học phục vụ nhu cầu học tập, nghiên cứu và làm việc hiệu quả | 158 Protein Purification Identification of critical amino acids involved in substrate hydrolysis Ferulic acid esterase features such as the catalytic triad and the oxyanion hole are usually maintained by several amino acid residues that are highly conserved among homologs. Other critical amino acids involved in substrate recognition and binding are also conserved among closely related homologs but not necessarily with less related homologs. A technique called alanine scanning or site-directed mutagenesis is helpful to determine the conserved amino acids critical for catalysis in proteins with unknown structure. The target amino acids selected for modification are replaced by alanine. Alanine is chosen because the inert alanine methyl functional group generally does not interact with other residues or alter the overall protein structure. To introduce the alanine mutation 39-nucleotide long complementary primers containing the desired amino acid replacement are used to introduce individual mutations. The protein variants are then constructed by Polymerase Chain Reaction using Finnymes PhusionTM high fidelity DNA polymerase. This approach was used to identify the critical amino acids of LJ0536 Lai et al. 2011 . The enzymatic activities of alanine variants are impared when the mutated amino acids are critical to function of the proteins. However the results obtained from alanine scanning may not be useful in distinguishing the specific function of the amino acids such as the involvement of amino acids in the formation of catalytic triad oxyanion hole tertiary structure of the protein or substrate recognition and binding. The amino acids involved in substrate recognition and binding can be determined by measuring the enzymatic activity of the protein variants with different substrate types. For example mutation of the amino acids that are only necessary for phenolic ester binding would not impair the enzymatic activity when aliphatic esters are used as substrates .

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