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Design and construction of a preparative-scale dynamic field gradient focusing apparatus.

ABSTRACT: A linear model is used to show that dynamic field gradient focusing (DFGF) can be scaled to preparative capacity, approximately O (10 mgs). This paper explains how the preparative-scale DFGF apparatus was designed and fabricated. Scaled-down experiments and mathematical modeling guided material selection and design changes during construction to increase the probability that the prototype preparative-scale DFGF apparatus would perform as intended. The finished prototype successfully focused bovine hemoglobin from an initial concentration of 6.82 to 15 mg/mL and allowed for 86% recovery of injected protein.

SUBMITTER: Tracy NI

PROVIDER: S-EPMC2925538 | biostudies-other | 2008 Mar-Apr

REPOSITORIES: biostudies-other

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Design and construction of a preparative-scale dynamic field gradient focusing apparatus.

Tracy Noah I NI Huang Zheng Z Ivory Cornelius F CF

Biotechnology progress 20080129 2

A linear model is used to show that dynamic field gradient focusing (DFGF) can be scaled to preparative capacity, approximately O (10 mgs). This paper explains how the preparative-scale DFGF apparatus was designed and fabricated. Scaled-down experiments and mathematical modeling guided material selection and design changes during construction to increase the probability that the prototype preparative-scale DFGF apparatus would perform as intended. The finished prototype successfully focused bovi ...[more]

PMID: 18225913

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Project description:Protein refolding is an important process to recover active recombinant proteins from inclusion bodies. Refolding by simple dilution, dialysis and on-column refolding methods are the most common techniques reported in the literature. However, the refolding process is time-consuming and laborious due to the variability of the behavior of each protein and requires a great deal of trial-and-error to achieve success. Hence, there is a need for automation to make the whole process as convenient as possible. In this study, we invented an automatic apparatus that integrated three refolding techniques: varying dilution, dialysis and on-column refolding. We demonstrated the effectiveness of this technology by varying the flow rates of the dilution buffer into the denatured protein and testing different refolding methods. We carried out different refolding methods on this apparatus: a combination of dilution and dialysis for human stromal cell-derived factor 1 (SDF-1/CXCL12) and thioredoxin fused-human artemin protein (Trx-ARTN); dilution refolding for thioredoxin fused-human insulin-like growth factor I protein (Trx-IGF1) and enhanced fluorescent protein (EGFP); and on-column refolding for bovine serum albumin (BSA). The protein refolding processes of these five proteins were preliminarily optimized using the slowly descending denaturants (or additives) method. Using this strategy of decreasing denaturants concentration, the efficiency of protein refolding was found to produce higher quantities of native protein. The standard refolding apparatus configuration can support different operations for different applications; it is not limited to simple dilution, dialysis and on-column refolding techniques. Refolding by slowly decreasing denaturants concentration, followed by concentration or purification on-column, may be a useful strategy for rapid and efficient recovery of active proteins from inclusion bodies. An automatic refolding apparatus employing this flexible strategy may provide a powerful tool for preparative scale protein production.

Dataset Information

Design and construction of a preparative-scale dynamic field gradient focusing apparatus.

Publications

Design and construction of a preparative-scale dynamic field gradient focusing apparatus.

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