The chromatography-free release, isolation and purification of recombinant peptide for fibril self-assembly.
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 cleavage 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 room temperature then removed contaminating proteins in an integrated isolation-purification step; it proved necessary to add 250 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.
Hartmann
B M
BM
Centre for Biomolecular Engineering, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia, QLD 4072, Australia.
Kaar
W
W
Yoo
I K
IK
Lua
L H L
LH
Falconer
R J
RJ
Middelberg
A P J
AP
D001709:Biotechnology / Q000379:methods*; D005591:Chemical Fractionation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics
D010455:Peptides; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D010447:Peptide Hydrolases