Indexed on: 12 Feb '08Published on: 12 Feb '08Published in: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]
This overview discusses issues involved with creating and manipulating vectors for expression of fusion proteins. The requirements for efficient translation include a promoter and a start codon, along with the fact that the mRNA encoding the protein to be expressed must contain a ribosome-binding site that is not blocked by mRNA secondary structure. The level of expression is also affected by codon preferences, and may be affected by the coding sequence in other ways that are not yet well understood. In virtually all cases, these problems can be solved by altering the sequence preceding the start codon, and/or by making changes in the 5' end of the coding sequence that do not change the protein sequence, taking advantage of the degeneracy of the genetic code. However, it is often quicker to solve these problems by making fusions between genes. In this approach the cloned gene is introduced into an expression vector 3' to a sequence (carrier sequence) coding for the amino terminus of a highly expressed protein (carrier protein). The carrier sequence provides the necessary signals for good expression, and the expressed fusion protein contains an N-terminal region encoded by the carrier. The carrier sequence can also code for an entire functional moiety or even for an entire protein that can be exploited in purifying the protein, either with antibodies or with an affinity purification specific for that carrier protein. Alternatively unique physical properties of the carrier protein (e.g., heat stability) can be exploited to allow selective purification of the fusion protein. Often, proteins fused to these carriers can be separated from the bulk of intracellular contaminants by taking advantage of special attributes.