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Chapter: Biotechnology Applying the Genetic Revolution: Recombinant Proteins

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Protein Fusion Expression Vectors

Joining the coding sequences of two proteins together in frame makes a protein fusion. Consequently, a single, longer polypeptide is made during translation.

PROTEIN FUSION EXPRESSION VECTORS


Joining the coding sequences of two proteins together in frame makes a protein fusion. Consequently, a single, longer polypeptide is made during translation. If the first (i.e., N-terminal) protein is normally secreted, then the fusion protein will be secreted, too.

Thus it is possible to achieve export of a recombinant protein by joining it to a protein that the cell normally exports.

 

Protein fusions also help address the issues of stability and purification. Many eukaryotic proteins are unstable inside the bacterial cell. This is especially true of growth factors, hormones, and regulatory peptides, which are often too short to fold into stable 3D configurations. Attaching them to the C terminus of a stable bacterial protein protects them from degradation. If the carrier protein is carefully chosen, purification may be greatly facilitated.

 

A protein fusion vector is a plasmid that allows the gene of interest to be fused to the gene for a suitable carrier protein. This is chosen so that it is an exported protein that is easy to purify. In addition, it should have a ribosomal binding site close to consensus and be translated efficiently. One of the best examples is the MalE protein of E. coli. This is a protein that is normally exported into the periplasmic space, where it transports maltose from the outer membrane to the inner membrane. Binding it to an amylose resin purifies MalE protein.

 

The gene of interest is cloned downstream and in frame with the MalE coding sequence. Between the coding sequences is a protease cleavage site, which allows the fused proteins to be cleaved apart after synthesis and purification. A strong promoter is also provided to maximize protein production. After protein expression, all the proteins are harvested from the periplasmic space. These are passed over an amylose column to bind the MalE fusion. The protein of interest is released from MalE and thus from the column by addition of the protease. Finally the protease is removed from the protein sample by another column that specifically binds protease.

 

A variety of other fusion protein systems have been used. We have already discussed the use of peptide tags, such as His6, FLAG, or GST to aid in protein purification. Here we are concerned with more sophisticated protein fusions that allow secretion of the fusion protein. Another effective fusion/secretion system uses the periplasmic enzyme β-lactamase. The β-lactamase domain binds to borate. This allows binding of the fusion protein (and the β-lactamase fragment after cleavage) by phenyl-boronate resins and the elution of the fusion protein from the resin with soluble borates.

 

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