In this work, we study the effect of the C-terminal 12x polyhistidine tag (His-tag), Recombinant as well as the peculiarities of the immobilized metal affinity chromatography (IMAC) protein purification procedure on the activity and structure of the metalloenzyme. l-alanyl-d-glutamate peptidase from bacteriophage T5 (EndoT5), whose zinc-binding site and catalytic aspartate are near the C-terminus. By itself, His-tag had no significant effect on either the activity or the folding of the polypeptide chain or the binding of zinc and calcium ions to the protein.
However, His-tagged EndoT5 samples had a low shelf life, and storage of these samples resulted in an increased propensity for protein self-association and decreased EndoT5 enzymatic activity. Furthermore, disastrous effects on enzyme activity were exerted by the presence of imidazole and nickel ions accompanying metal chelate chromatography. Protein activity can be restored by thoroughly washing out these low molecular weight impurities by prolonged dialysis of His-tagged EndoT5 samples under specifically made conditions.
Keywords: Conformational stability; Enzymatic activity; Your label; structural properties; zinc bonding; l-alanyl-d-glutamate peptidase
The length of your label influences the yield and purity of the IMAC column.
From time to time, we received questions such as: why is 6xHis (6 constitutive histidine residues) used in most recombinant proteins that are constructed for IMAC purification? How about 4xHis or 10xHis? How does polyHis tag length affect IMAC purification?
It is true that the most widely used polys tag consists of 6 constitutive histidine residues. Longer or shorter polyHis tags have been used very infrequently, but have been shown to be effective in purifying various target proteins. The reason for using 6xHis could be determined through trial and error, as it is the shortest stretch of polyhistidine peptide that can be purified by IMAC to satisfactory purity in high yield.
In early one-step purification of polys-tagged target proteins by IMAC, 6xHis was successfully used to purify 95% pure recombinant protein constructs in high yield. Importantly, the 6xHis tag appeared not to disrupt the functions and activities of tagged proteins from various expression hosts.
How polyHis tag length influences purification in IMAC
The length of the polyHis tag can influence the performance of purification on the IMAC column, both in terms of yield and purity. The multiplicity of histidine residues in a protein is associated with its affinity for immobilized metal ions. Two histidine residues are enough to bind to the Ni-IDA backbone, which has a very high binding affinity for histidine. However, a pair of adjacent histidines bind weakly to the Ni-NTA column and will be washed from the column by washing buffers with low concentrations of imidazole. As the number of histidine residues in the stretch increases, the binding to the IMAC column becomes stronger. Therefore, it requires a larger amount of imidazole to elute from the column.
Since a larger number of histidine residues results in a higher binding affinity, by extending the polyhistidine tag, the target fusion protein can bind to IMAC columns more efficiently and more strongly, even if it is expressed at a higher level, low level. In practice, the long His-tag is also advantageous in achieving higher purity, because it allows more stringent washes at a high concentration of imidazole to remove non-specifically bound contaminants. These advantages become more prominent in applications using highly selective columns, such as Co-NTA and Ni-Penta columns. 10xHis was first successfully used to purify a recombinant neurotensin receptor, which was expressed at very low levels in E.coli. 10xHis was able to achieve a 340-fold enrichment of functional receptor to 72% purity on Cobalt NTA columns.
When to consider a longer polyHis tag
Since the 6xHis tag has been shown to be effective in purifying many target proteins, it is recommended to always start with 6xHis when constructing a new protein. For many proteins, purification can be improved to satisfaction simply by adjusting the purification conditions. However, when there is little or no improvement after all optimization efforts have been made, the idea of adapting the fusion protein construct may occur to you. Here are some situations where longer polyHis tags (i.e. 8xHis or 10xHis) might be useful to improve the yield and purity of the target protein if used:
- Impurity or contaminating proteins are persistently “co-purified” with the target protein.
Some strains of E.coli contain histidine-rich proteins, which can adhere to the IMAC column. For example, a protein from E. coli consisting of a domain homologous to FKBP has been detected as a persistent contaminant in IMAC. In many mammalian systems, there is a higher natural abundance of proteins containing consecutive histidine residues that lead to specific binding in IMAC. For example, it was found in nuclear extracts from undifferentiated HeLa cells that OCT6 can be easily purified on a NiNTA column due to its natural stretch of 6-histidine near the C-terminus, as well as another His-rich sequence.
In another study, TGF-β1 was found to be NiNTA purified from HEK293 cell culture media using a standard protocol. Disulfide bond formation between the protein of interest and other proteins can also lead to contamination. Furthermore, non-specific hydrophobic interactions may cause some co-purification with the desired protein. In these cases, more stringent binding and washing conditions may be necessary to minimize persistent contaminations. By expanding the number of histidine residues in the fusion tag, it will increase the binding of the fusion construct in IMAC, allowing the use of higher imidazole concentrations during purification steps.
- Target protein does not bind well to highly selective IMAC resins
The selectivity and affinity of IMAC for the His-tag are two sides of the same coin. Highly selective IMAC resins (such as Co-NTA and Ni-Penta) have a low affinity for the polyHis tag. In some cases where target protein expression is low, capturing the protein on highly selective IMAC columns becomes challenging. Increasing the number of histidine on the tag can help improve the affinity of the tag for immobilized metal ions and thus can increase the yield after purification.
Facts to note for using the long polyHis tag
We may have to say that the impact of polyHis length on the target protein sometimes goes beyond its behaviour on the IMAC column. Here are some of the potential issues to be aware of when trying to tailor the length of the polyHis tag for better purity and performance:
- The longer polyHis chain is more flexible, so it could reach specific sites that are involved in protein functions, inhibiting protein activities. Removing the label may be necessary for further analysis.
- The flexible long-chain could also induce conformational changes of the fusion protein construct by interacting with non-specific sites, increasing the oligomeric state of the protein. This is particularly the case for membrane proteins. Consequently, the fusion protein forms aggregates and is depleted in the soluble fraction.
- The expression level of the fusion protein in the host may also be affected. It was suggested that the length of the polyHis tag has a greater negative impact on protein expression than the position of the tag.
- In another aspect, as the longer polyHis tag has a greater affinity for the immobilized metal, a greater amount of imidazole will be required to elute the bound target protein from the column. For example, in the purification of the 10xHis-tagged recombinant AqpZ protein fused in Co-NTA, 500 mM imidazole was needed to elute the 10xHis-tagged AqpZ protein, rather than 100 mM imidazole which was sufficient for elution of the 10xHis-tagged AqpZ protein. tagged with 6xHis. However, exposure to a high concentration of imidazole is undesirable for some target proteins, as they are sensitive to imidazole and can become unstable in solutions containing a large amount of imidazole. It is advisable to keep the amount of imidazole as low as possible throughout the entire protocol.