Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Fundamentals of Molecular Biotechnology
Optimization of Gene Expression Cloned in Prokaryotic Systems
Protein Stabilization

Typically, the half-life of Proteins ranges from a few minutes to several hours. This Variability is governed by differences in the number of Disulfide Bonds within the protein molecules and the presence or absence of specific Amino Acids at the 5'-end. For instance, attaching various amino acids to the N-terminus of ß-galactosidase can cause the in vitro half-life of the modified protein to range from two minutes to over 20 hours (Table 6.4). Amino acids that extend protein half-life can be incorporated into proteins using Introduction/32.html">Genetic Engineering techniques. Often, attaching just a single amino acid residue to the N-terminus is sufficient to stabilize a target protein. Long-lived proteins accumulate within Cells, thereby increasing the final product yield. This phenomenon is characteristic of both eukaryotes and prokaryotes.

Class="center">Table 6.4. Half-life of ß-galactosidases with various amino acids attached to their N-terminus¹⁾

¹⁾ Based on Bachmair et аl., 1986, Science 234: 179—186.

However, protein stability can also be decreased rather than enhanced. For example, incorporating certain Amino acid sequences into the interior of a protein molecule makes it more susceptible to proteolytic Cleavage. These sequences are enriched in Proline (P), glutamic acid (E), Serine (S), and Threonine (T) residues, hence their designation as PEST sequences. They are frequently flanked by clusters of positively charged Amino Acids and likely act as degradation signals (destabilizing tags) for proteases. The stability of proteins containing such sequences could theoretically be improved by engineering the corresponding genes. Care must be taken, however, to ensure that these modifications do not disrupt the function of the target protein.