The Chromatography-Free Release, Isolation and Purification of Recombinant Peptide for Fibril Self-Assembly

Hartmann, B. M., Kaar, W., Yoo, I. K., Lua, L. H. L., Falconer, R. J. and Middelberg, A. P. J. (2009) The Chromatography-Free Release, Isolation and Purification of Recombinant Peptide for Fibril Self-Assembly. Biotechnology and Bioengineering, 104 5: 973-985. doi:10.1002/bit.22447


Author Hartmann, B. M.
Kaar, W.
Yoo, I. K.
Lua, L. H. L.
Falconer, R. J.
Middelberg, A. P. J.
Title The Chromatography-Free Release, Isolation and Purification of Recombinant Peptide for Fibril Self-Assembly
Journal name Biotechnology and Bioengineering   Check publisher's open access policy
ISSN 0006-3592
Publication date 2009-12-01
Year available 2009
Sub-type Article (original research)
DOI 10.1002/bit.22447
Volume 104
Issue 5
Start page 973
End page 985
Total pages 13
Editor Douglas S. Clark
Place of publication United States
Publisher John Wiley & Sons
Collection year 2010
Language eng
Subject C1
Formatted abstract
One of the major expenses associated with recombinant peptide production is the use of chromatography in the isolation and purification stages of a bioprocess. Here we report a chromatography free isolation and purification process for recombinant peptide expressed in Escherichia coli (E. coli). Initial peptide release is by homogenization and then by enzymatic clevage of the peptide-containing fusion protein, directly in the E. coli homogenate. Release is followed by selective solvent precipitation (SSP) to isolate and purify the peptide away from larger cell contaminants. Specifically, we expressed in E. coli the self-assembling beta-sheet forming peptide P-11-2 in fusion to thioredoxin. Homogenate was heat treated (55 degrees C, 15 min) and then incubated with tobacco etch virus protease (TEVp) to release P-11-2 having a native N-terminus. SSP with ethanol at a room temperature then removed contaminating proteins in an integrated isolation purification step; it proved necessary to add 205 mM NaCl to homogenate to prevent P-11-2 from partitioning to the precipitate. This process structure gave recombinant P-11-2 peptide at 97% polypeptide purity and 40% overall yield, without a single chromatography step. Following buffer exchange of the 97% pure product by bind-elute chromatography into defined chemical conditions, the resulting peptide was shown to be functionally active and able to form self-assembled fibrils. To the best of our knowledge, this manuscript reports the first published process for chromatography free recombinant peptide release, isolation and purification. The process proved able to deliver functional recombinant peptide at high purity and potentially low cost, opening cost-sensitive materials applications for peptide-based materials. Biotechnol. Bioeng. 2009;104: 973-985. © 2009 Wiley Periodicals, Inc.
Keyword peptide
fibril
cost
recombinant
biomaterials
chromatography-free
HIGH-LEVEL EXPRESSION
ETCH VIRUS PROTEASE
BETA-SHEET PEPTIDES
ESCHERICHIA-COLI
ANTIMICROBIAL PEPTIDE
FUSION EXPRESSION
TEV PROTEASE
ANTIBACTERIAL PEPTIDE
MECHANICAL-PROPERTIES
INCLUSION-BODIES
Q-Index Code C1
Q-Index Status Confirmed Code

Document type: Journal Article
Sub-type: Article (original research)
Collections: 2010 Higher Education Research Data Collection
Australian Institute for Bioengineering and Nanotechnology Publications
 
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Citation counts: TR Web of Science Citation Count  Cited 13 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 13 times in Scopus Article | Citations
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Created: Sun, 29 Nov 2009, 00:05:33 EST