Promiscuity of host cell proteins in the purification of histidine tagged recombinant xylanase A by IMAC procedures: A case study with a Ni2+-tacn-based IMAC system
| Autor: | Yuanzhong Yang, Reinhard I Boysen, Khosse Mitri, Chunfang Zhang, Milton T.W. Hearn |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
0303 health sciences Chromatography biology Elution Nitrilotriacetic acid Tandem mass spectrometry biology.organism_classification medicine.disease_cause 01 natural sciences Sepharose 03 medical and health sciences chemistry.chemical_compound chemistry 13. Climate action 010608 biotechnology medicine bacteria Agarose Bacillus halodurans Escherichia coli Histidine 030304 developmental biology Biotechnology |
| Zdroj: | Protein Expression and Purification. 162:51-61 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2019.05.009 |
| Popis: | Determination of the extent of host cell protein (HCP) contamination is an essential pre-requisite to validate the chromatographic purification of recombinant proteins. This study explores how different experimental conditions affect the HCP profiles generated during the immobilised metal ion affinity chromatographic (IMAC) purification with a Ni2+-1,4,7-triaza-cyclononane (tacn) Sepharose FF™ sorbent of the Bacillus halodurans N- and C-terminal His6-tagged xylanase A, expressed by Escherichia coli BL21(DE3) cells, and captured directly from cell lysates. Comparative studies were also carried out under identical loading, wash and elution conditions using nitrilotriacetic acid (NTA), also immobilised onto an agarose support and complexed with Ni2+ ions. High-resolution tandem mass spectrometry of the tryptic peptides derived from the proteins present in the IMAC flow-through, wash and elution fractions confirmed that the E. coli BL21(DE3) HCP profiles were dependent on the choice of adsorbent. With feedstocks containing the N- or C-terminal His6-tagged xylanase A, in several instances the same E. coli BL21(DE3) HCPs were found to co-elute with the tagged protein from either adsorbent, indicating a preferential ability of some HCPs to bind to both the IMAC resin and to the recombinant protein. This promiscuous behaviour has been found to be due to factors other than just the presence of histidine-rich motifs within the amino acid sequences of these HCPs. This case study demonstrates that the choice of protein expression and separation conditions impact on the levels of HCP contamination when different IMAC systems are employed. |
| Databáze: | OpenAIRE |
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