Abstract
“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.
Highlights
The Holy Grail for the biotech industry of today is to achieve optimal and robust manufacturing processes that are economic and sus tainable in a time- and resource-efficient manner
After assessing the buffering and incubation conditions for maximum binding capacity and desorption efficiency, we explored the use of bare silica particles as matrix for affinity purification of His6-tagged proteins from clarified E. coli extracts
We demonstrated that the His6 tag has silica-binding prop erties and that bare silica can be used as affinity matrix to purify Histagged proteins from bacterial cell extracts with high yield and purity
Summary
The Holy Grail for the biotech industry of today is to achieve optimal and robust manufacturing processes that are economic and sus tainable in a time- and resource-efficient manner. Owing their demands, the processes for production and purification of recombinant proteins hit their goal when high yields of the target protein are achieved while maintaining high levels of purity and activity [1,2]. Superficial silanol groups turn its surface very versatile towards surface chemistry modifications for a variety of ap plications in the biochemistry and biotechnology scientific domains [7]
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