VPB 321: Animal Biotechnology (2+1)

EXPRESSION VECTORS THAT PRODUCE FUSION PROTEINS

• When most expression vectors operate, they produce fusion proteins.  This might at first seem a disadvantage because the natural product of the inserted gene is not made.  However, the extra amino acids on the fusion protein can be a great help in purifying the protein product.
• Consider the oligo-histidine expression vectors, one of which has the trade name pTrcHis.  These have a short sequence just upstream of the multiple cloning site that encodes a stretch of six histidines. 
• Thus, a protein expressed in such a vector will be a fusion protein with six histidines at its amino end. 
• The advantage of this, is oligo-histidine regions like this have a high affinity for metals like nickel, so the expressed proteins can be purified by using nickel affinity chromatography. 
• After the bacteria have made the fusion protein, simply lyse them, add the crude bacterial extract to a nickel affinity column, wash out all unbound proteins, then release the fusion protein with histidine or a histidine analog called imidazole. 
• This procedure allows to harvest essentially pure fusion in only one step. 
• This is possible because very few if any natural proteins have oligo-histidine regions, so the fusion protein is essentially the only one that binds to the column.