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Techniques to Interrogate Active Site Interactions
Biomolecules
University of Lincoln
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Techniques to interrogate active site interactions
● Plasmids ○ Used to transfer genetic information from one cell to another ○ Range in size from ~3kb - 100s of kb ○ Circular and double stranded DNA ○ Separate from chromosomal DNA ○ Are replicated along with the hosts chromosomal DNA during cell division ○ Occur naturally in bacteria, yeast and some higher eukaryotes ○ Most contain genes beneficial to the host cells
● Traditional cloning ○ Uses restriction enzymes ■ Kessler and Manta (1990) specificity of restriction endonucleases and DNA modification methyltransferase ○ Restriction enzymes recognise and cut specific sequences ~4 in double stranded DNA ○ Recognition sequences are palindromic
● Expression vectors ○ In addition to the ORI and marker genes for E, expression vectors have additional components depending on their target host ○ Marker genes include: ■ Growth-based markers in yeast e. expression of enzymes involved in producing essential amino acids ■ Herbicide resistance markers for selection in plants ○ If you wish to express a protein a promoter must be present to allow transcription ■ Promotors can be constitutive or inducible ○ Terminators are also present to terminate transcription ○ pET-28b expression vector allows the expression of tagged N or C-terminally tagged protein
● Traditional cloning 1. Map of ORF and select MCS sites that are not present
● Preparation of insert
Amplify the ORF using PCR with flanking compatible restriction sites
Produce ‘sticky ends’ on the ORF
● Preparation of vector
● Cut the vector with compatible sites ● Gel purify to remove fragments of the digest ● Ligate the ORF in to the Vector using DNA ligase ○ Note only one orientation when different sites are used
○ Multiple sequence alignment ■ Clustal Omega is a alignment program that uses seeded guide tress and HMM profile-profile techniques to generate alignments between three or more sequences
● Use your clustal alignment to spot a conserve Y and map it onto the translated protein
● The effect of mutagenesis on enzyme activity ○ What do you think the activity of Y253A mutation would be if it is a catalytic residue? ○ What do you think the activity of Y253A mutation would be if it is not a catalytic residue?
Techniques to Interrogate Active Site Interactions
Module: Biomolecules
University: University of Lincoln
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- More from:BiomoleculesUniversity of Lincoln33 Documents
