Publicação
Bare silica as an alternative matrix for affinity purification/immobilization of His-tagged proteins
| Resumo: | Green protein purification/immobilization processes based on low-cost, earth-abundant, and eco-friendly affinity matrices are highly desirable. Unmodified silica matrices fit well these demands. Since histidine-rich silica-binding peptides are frequently isolated in biopanning experiments, this work aimed at assessing the viability of using bare silica as an alternative matrix for the purification/immobilization of His-tagged proteins. Adsorption and desorption studies with a purified His6-tagged EGFP shown that binding to bare silica particles of different size and porosity occurred under the conditions tested, and that elution could be accomplished with eco-friendly eluants containing L-arginine/L-lysine. Non-tagged EGFP did not bind to these matrices. Small-scale batch purification schemes using silica gel Davisil grade 643 or 646 as affinity matrices and a Tris-buffered saline eluant containing 0.5 M L-arginine (pH 8.5) allowed purifying His6-EGFP from Escherichia coli lysates with a purity of up to 96 % and a recovery yield of 70 % after just one elution step. EGFP tagged with the silica-binding peptide Car9 was recovered with comparable purity and yield. Other His-tagged proteins could also be purified to similar purity levels. The scale of this batch purification scheme was shown to be extendable. These results demonstrate that unmodified silica matrices can be used to effectively purify His-tagged proteins. Since the recovery of double tagged His6-EGFP-Car9 was only of 30-55 %, the combination of tags revealed to be advantageous for immobilization purposes. |
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| Autores principais: | Freitas, Ana Isabel |
| Outros Autores: | Domingues, Lucília; Aguiar, Tatiana Quinta |
| Assunto: | Silicon dioxide Silica-binding peptides His-tag Affinity purification/immobilization Batch affinity chromatography Affinity purification immobilization |
| Ano: | 2022 |
| País: | Portugal |
| Tipo de documento: | artigo |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Green protein purification/immobilization processes based on low-cost, earth-abundant, and eco-friendly affinity matrices are highly desirable. Unmodified silica matrices fit well these demands. Since histidine-rich silica-binding peptides are frequently isolated in biopanning experiments, this work aimed at assessing the viability of using bare silica as an alternative matrix for the purification/immobilization of His-tagged proteins. Adsorption and desorption studies with a purified His6-tagged EGFP shown that binding to bare silica particles of different size and porosity occurred under the conditions tested, and that elution could be accomplished with eco-friendly eluants containing L-arginine/L-lysine. Non-tagged EGFP did not bind to these matrices. Small-scale batch purification schemes using silica gel Davisil grade 643 or 646 as affinity matrices and a Tris-buffered saline eluant containing 0.5 M L-arginine (pH 8.5) allowed purifying His6-EGFP from Escherichia coli lysates with a purity of up to 96 % and a recovery yield of 70 % after just one elution step. EGFP tagged with the silica-binding peptide Car9 was recovered with comparable purity and yield. Other His-tagged proteins could also be purified to similar purity levels. The scale of this batch purification scheme was shown to be extendable. These results demonstrate that unmodified silica matrices can be used to effectively purify His-tagged proteins. Since the recovery of double tagged His6-EGFP-Car9 was only of 30-55 %, the combination of tags revealed to be advantageous for immobilization purposes. |
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