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A general strategy for the bacterial expression of amyloidogenic peptides using BCL-XL-1/2 fusions.

ABSTRACT: 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.

SUBMITTER: Yonemoto IT

PROVIDER: S-EPMC2777372 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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A general strategy for the bacterial expression of amyloidogenic peptides using BCL-XL-1/2 fusions.

Yonemoto Isaac T IT Wood Malcolm R MR Balch William E WE Kelly Jeffery W JW

Protein science : a publication of the Protein Society 20090901 9

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 lo ...[more]

PMID: 19621381

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Project description:BCL-xL and BCL-2 are validated therapeutic targets in small-cell lung cancer (SCLC). Targeting these proteins with navitoclax (formerly ABT263, a dual BCL-xL/2 inhibitor) induces dose-limiting thrombocytopenia through on-target BCL-xL inhibition in platelets. Therefore, platelet toxicity poses a barrier in advancing the clinical translation of navitoclax. We have developed a strategy to selectively target BCL-xL in tumors, while sparing platelets, by utilizing proteolysis-targeting chimeras (PROTACs) that hijack the cellular ubiquitin proteasome system for target ubiquitination and subsequent degradation. In our previous study, the first-in-class BCL-xL PROTAC, called DT2216, was shown to have synergistic antitumor activities when combined with venetoclax (formerly ABT199, BCL-2-selective inhibitor) in a BCL-xL/2 co-dependent SCLC cell line, NCI-H146 (hereafter referred to as H146), in vitro and in a xenograft model. Guided by these findings, we evaluated our newly developed BCL-xL/2 dual degrader, called 753b, in three BCL-xL/2 co-dependent SCLC cell lines and the H146 xenograft models. 753b was found to degrade both BCL-xL and BCL-2 in these cell lines. Importantly, it was considerably more potent than DT2216, navitoclax, or DT2216 + venetoclax in reducing the viability of BCL-xL/2 co-dependent SCLC cell lines in cell culture. In vivo, 5 mg/kg weekly dosing of 753b was found to lead to significant tumor growth delay, similar to the DT2216 + venetoclax combination in H146 xenografts, by degrading both BCL-xL and BCL-2. Additionally, 753b administration at 5 mg/kg every four days induced tumor regressions. At this dosage, 753b was well tolerated in mice, without observable induction of severe thrombocytopenia as seen with navitoclax, and no evidence of significant changes in mouse body weights. These results suggest that the BCL-xL/2 dual degrader could be an effective and safe therapeutic for a subset of SCLC patients, warranting clinical trials in future.

Dataset Information

A general strategy for the bacterial expression of amyloidogenic peptides using BCL-XL-1/2 fusions.

Publications

A general strategy for the bacterial expression of amyloidogenic peptides using BCL-XL-1/2 fusions.

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