Cloning technologies for protein expression and purification(1)
Protein overexpression requires three components: a gene, a vector that contains the gene, and an expression host that maximizes the amount and quality of the protein produced by the vector–gene combination. Expression in Escherichia coli is fast, inexpensive and scaleable, and minimal post-translational modifications make proteins purified from E. coli relatively homogeneous and highly desirable for structural studies. However, most eukaryotic proteins larger than 30 kDa are unlikely to be properly folded when expressed in E. coli. Expression in mamma- lian cells is best for activity and native structure (including post-translational modifications), but yields are much lower and costs are high. Using a cDNA as a PCR template, the gene of interest is typically amplified (sometimes in multiple versions with various modifications), cloned, sequenced, and then sub- cloned into one or more expression vectors.
The sequences commonly can be added to genes that affect translation of the gene or which encode domains that make the resulting protein more useful or easier to purify.
Many of these motifs, along with cloning sites, are added to PCR primers and are thus incorporated into PCR products.
Additional protein expression elements are found in the vectors into which gene cassettes are introduced.
There are the primary cloning methods used in the protein expression literature.
Most protein expression plasmids are constructed with restriction enzymes and ligase (REaL). a vector is acquired and the gene is designed to be compatible with the vector (i.e. appropriate restriction sites, tags, reading frame, orienta- tion), usually as part of a PCR amplification step. The multitude of protein expression vectors offers a universe of solutions to cloning, expression and purification problems, but their diversity, in both use and structure, makes acquiring and using them a tedious process.
Recommendations for choosing the best cloning and expression strategies are summarized.
