Protein stability is another common problem in protein expression. It is also an important topic in purification, formulation, and storage. Here we will discuss protein stability in expression only. Properly folded proteins are usually stable during expression and purification. Sufficient amount of intact protein should be obtained.
However, some proteins appear to be unstable during expression and purification. Some of them are so unstable that sufficient amount of protein cannot be obtained.
Basic points on Protein Stability:
Many factors such as an amino acid sequence of the protein, protein construction, host cell strain, expression and purification conditions may affect protein stability.
- Amino acid sequence of a protein itself may be susceptible to degradation. Certain amino acids at the N-terminus of a protein can lead the protein to degradation. These are Arg, Lys, Leu, Phe, Tyr, and Trp residues. Replacing these amino acids with others can greatly increase the protein half-life (N-end rule).
- Many recombinant proteins are expressed by tags or fusion partners. Amino acid sequences at N-termini of these tags and fusion partners are often optimized for protein yield and stability. Therefore amino acids at N-terminus are not a problem in protein stability for these tagged or fusion proteins.
- It is reported that regions containing Pro (P), Glu (E), Ser (S), and Thr (T) termed PEST are prone to degradation. It is generally observed that flexible hydrophilic sequences with protease cleavage sites are easily degraded.
- These sequences may be an integral part of a protein. In most cases, these sequences cannot be deleted or mutated. Strategies for improving protein stability are needed for these proteins.
Strategies to improve Protein Stability
[su_list icon=”icon: arrow-right” icon_color=”#007213″]
Perform expression in special media containing trace metals, minerals, and vitamins. These chemicals may not be needed for host cell growth, but they may serve as a cofactor, prosthetic groups or ligands for recombinant proteins. Therefore they may be critical for correct protein folding and stability. Medium pH should also be balanced near neutral to improve protein stability. There will be no protein degradation caused by nutrition exhaustion in our special media.
Induce the protein at a lower temperature and/or for shorter induction time.
Fuse the protein with a tag or fusion partner. A tag can change the N-terminal sequence of the protein and therefore increase the yield and stability. In addition to N-terminal sequence change, a relative large fusion partner can further stabilize the protein compared with that the protein was expressed alone or with a small tag.
Design the protein construct with intact domain or structure. A full-length protein, a part of a protein with intact domains, or an intact domain of a protein can be stably expressed. An integral folding unit of a protein cannot be truncated. Otherwise, it may not be structured and therefore will not be stable. A domain is often a folding unit. Truncations at either terminus may disrupt protein folding. A domain of a protein can be determined by homologous alignment of the protein with other proteins.
The more proteins are used in the alignment, the more accurate boundaries of a domain can be determined. Including more amino acids at the boundaries is often better than trimming off some residues. If an intact domain has to be truncated, fusing it with a large partner such as GST will make it more stable. Small tags such as his-tag will not improve protein stability in these cases.
Change the host cell strain. Some cell strains are deficient in some proteases. For example, BL21 lacks cytoplasmic ion and periplasmic ompT proteases. Using these cell strains will lead to enhanced protein stability. Sometimes simply changing a host strain will increase recombinant protein stability.
Change the location of expression. Some proteins are not stable if they are expressed in the cytoplasm. When they are expressed in the periplasmic region, it becomes stable. The periplasmic expression may lead to correct folding of a protein. The periplasmic region may also lack the protease to degrade the protein.
Express the protein in cell strains containing molecular chaperones. Molecular chaperones may facilitate protein folding and increase it stability
It is clear that strategies such as using special media, cell strains or growth conditions are easy to implement. Making fusion protein, designing protein construct, and changing expression location will involve DNA manipulation. In the case that a truncated protein domain is unstable, the choices may be to re-design the construct and express the protein as an intact domain or to fuse it with large protein partner.
[su_note note_color=”#f5ffde” text_color=”#000000″ radius=”0″][su_posts template=”templates/list-loop.php” posts_per_page=”5″ tax_term=”7″ tax_operator=”0″ order=”asc” orderby=”rand”][/su_note]