Bioprocess-centered molecular design (BMD) for the efficient production of an interfacially active peptide.
The efficient expression and purification of an interfacially active peptide (mLac21) was achieved by using bioprocess-centered molecular design (BMD), wherein key bioprocess considerations are addressed during the initial molecular biology work. The 21 amino acid mLac21 peptide sequence is derived from the lac repressor protein and is shown to have high affinity for the oil-water interface, causing a substantial reduction in interfacial tension following adsorption. The DNA coding for the peptide sequence was cloned into a modified pET-31(b) vector to permit the expression of mLac21 as a fusion to ketosteroid isomerase (KSI). Rational iterative molecular design, taking into account the need for a scaleable bioprocess flowsheet, led to a simple and efficient bioprocess yielding mLac21 at 86% purity following ion exchange chromatography (and >98% following chromatographic polishing). This case study demonstrates that it is possible to produce acceptably pure peptide for potential commodity applications using common scaleable bioprocess unit operations. Moreover, it is shown that BMD is a powerful strategy that can be deployed to reduce bioseparation complexity.
Morreale
Giacomo
G
Department of Chemical Engineering, University of Cambridge, Pembroke Street, CB2 3RA, UK.
Lee
Eun Gyo
EG
Jones
Daniel B
DB
Middelberg
Anton P J
AP
D003001:Cloning, Molecular / Q000379:methods; D015195:Drug Design; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods*; D008967:Molecular Biology / Q000379:methods; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013500:Surface Tension; D013501:Surface-Active Agents / Q000737:chemistry / Q000378:metabolism*
D010455:Peptides; D011994:Recombinant Proteins; D013501:Surface-Active Agents