A general strategy for the bacterial expression of amyloidogenic peptides using BCL-XL-1/2 fusions.
Biophysical studies on amyloidogenic and aggregation-prone peptides often require large quantities of material. However, solid-phase synthesis, handling, and purification of peptides often present challenges on these scales. Recombinant expression is an attractive alternative because of its low cost, the ability to isotopically label the peptides, and access to sequences exceeding approximately 50 residues. However, expression systems that seek to solubilize amyloidogenic peptides suffer from low yields, difficult optimizations, and isolation challenges. We present a general strategy for expressing and isolating amyloidogenic peptides in Escherichia coli by fusion to a polypeptide that drives the expression of attached peptides into bacterial inclusion bodies. This scheme minimizes toxicity during bacterial growth and enables the processing and handling of the peptides in denaturing solutions. Immobilized metal affinity chromatography, reverse phase HPLC, and cyanogen bromide cleavage are used to isolate the peptide, followed by further reverse phase HPLC, which yields milligram quantities of the purified peptide. We demonstrate that driving the peptides into inclusion bodies using fusion to BCL-XL-1/2 is a general strategy for their expression and isolation, as exemplified by the production of 11 peptides species.
Yonemoto
Isaac T
IT
Department of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Wood
Malcolm R
MR
Balch
William E
WE
Kelly
Jeffery W
JW
D000595:Amino Acid Sequence; D000682:Amyloid / Q000235:genetics* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D002479:Inclusion Bodies / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics* / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D051020:bcl-X Protein / Q000235:genetics* / Q000302:isolation & purification
D000682:Amyloid; D010455:Peptides; D011993:Recombinant Fusion Proteins; D051020:bcl-X Protein