iGenetics 3rd Edition by Peter Russell

Exercise 7

One frequent objective of expressing a protein in E. coli using an expression vector is to purify it. If the expressed protein is "tagged" at one end with a particular peptide sequence, it is easier to purify. For example, proteins tagged at one end with six histidine residues can be recovered from lysed E. coli cells by incubating the lysate with a nickel-containing resin. The six-histidine tag has a high affinity for the resin, facilitating the purification of the protein from the lysate. Proteins tagged in this way are fusion proteins-they contain the amino acid sequence encoded by an open reading frame (ORF) of a cDNA fused to the amino acid sequence of the tag. Some plasmid vectors have been designed to facilitate the production of such fusion proteins. They have an E. coli promoter sequence for transcription initiation and are designed so that the RNA that is produced will have a Shine-Dalgarno sequence near its 5? end to facilitate ribosome binding. Following this, they have an ORF with the codons for the tag at its end. A multiple cloning site (MCS) is embedded within the ORF to facilitate cloning of part of a cDNA. Figure shows the MCS in one such vector. In the figure, The 5? -to- 3? DNA strand that has the same polarity as the resulting mRNA is in bold type, the amino acids it encodes are given underneath their codons, and three unique restriction enzyme sites in the vector are shadowed in grey with the sites of DNA cleavage indicated by lines.
a. Suppose your want to tag a polypeptide encoded by a cloned cDNA whose ORF includes Xho I and Eco RI sites close to its beginning and a Pst I site close to its end. What steps would you take to insert a fragment containing most of the cDNA's ORF into this expression vector? Can you be certain that a fusion protein will be produced?
b. How would you clone the ORF of a previously cloned cDNA into this expression vector if the cDNA had no Xho I, Pst I, or Eco RI sites? What concerns would you need to address to ensure that a fusion protein would be produced?
Figure