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Recombinant protein purification using complementary peptides as affinity tags

Paper ID Volume ID Publish Year Pages File Format Full-Text
33438 44978 2012 5 PDF Available
Title
Recombinant protein purification using complementary peptides as affinity tags
Abstract

Affinity tags have become highly popular tools for purifying recombinant proteins from crude extracts by affinity chromatography. Besides, short peptides are excellent ligands for affinity chromatography, as they are not likely to cause an immune response in case of leakage into the product, they are more stable than antibodies to elution and cleaning conditions and they usually have very acceptable selectivity. Hydropathically complementary peptides designed de novo show enough selectivity to be used successfully as peptide ligands for protein purification from crude extracts. Recognition specificity and selectivity in the interaction between the complementary peptide pair His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu and Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe have been demonstrated by other authors.In this work, we designed a recombinant protein purification method using a peptide affinity tag that binds to a peptide-binding partner immobilized on a chromatographic matrix. The enhanced green fluorescent protein expressed (EGFP) in Escherichia coli was used as the model.The peptide Gly-Gly-Gly-His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu was synthesized by solid phase using the Fmoc chemistry and immobilized in NHS-Sepharose (PC-Sepharose). Gly residues were added as a spacer arm at the N terminus. The EGFP was expressed either with the fusion tag Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe on the C terminus (EGFP-CPTag) or without any fusion tag.After cell disruption, the extract was directly applied to the PC-Sepharose column equilibrated with 20 mM sodium phosphate buffer, pH 7.0. The adsorbed EGFP-CPTag was then eluted with 1 M Tris. The yield was 98% and the purification factor 4.6. By contrast, EGFP without tag pass through without interacting with the PC-Sepharose column.The method designed can be applied for the purification of other recombinant proteins.

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Recombinant protein purification using complementary peptides as affinity tags
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Publisher
Database: Elsevier - ScienceDirect
Journal: New Biotechnology - Volume 29, Issue 2, 15 January 2012, Pages 206–210
Authors
, , , , , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
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Full-text PDF Download
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