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Expanding the utility of beta-galactosidase complementation: piece by piece.

ABSTRACT: The ability to image and quantify multiple biomarkers in disease necessitates the development of split reporter fragment platforms. We have divided the beta-galactosidase enzyme into unique, independent polypeptides that are able to reassemble and complement enzymatic activity in bacteria and in mammalian cells. We created two sets of complementing pairs that individually have no enzymatic activity. However, when brought into close geometric proximity, the complementing pairs associated resulting in detectable enzymatic activity. We then constructed a stable ligand complex composed of reporter fragment, linker, and targeting moiety. The targeting moiety, in this case a ligand, allowed cell surface receptor targeting in vitro. Further, we were able to simultaneously visualize two cell surface receptors implicated in cancer development, epidermal growth factor receptor and transferrin receptor, using complementing pairs of the ligand-reporter fragment complex.

SUBMITTER: Broome AM

PROVIDER: S-EPMC2835542 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Expanding the utility of beta-galactosidase complementation: piece by piece.

Broome Ann-Marie AM Bhavsar Nihir N Ramamurthy Gopalakrishnan G Newton Gail G Basilion James P JP

Molecular pharmaceutics 20100201 1

The ability to image and quantify multiple biomarkers in disease necessitates the development of split reporter fragment platforms. We have divided the beta-galactosidase enzyme into unique, independent polypeptides that are able to reassemble and complement enzymatic activity in bacteria and in mammalian cells. We created two sets of complementing pairs that individually have no enzymatic activity. However, when brought into close geometric proximity, the complementing pairs associated resultin ...[more]

PMID: 19899815

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Project description:BACKGROUND: Antibody fragments are molecules widely used for diagnosis and therapy. A large amount of protein is frequently required for such applications. New approaches using folding reporter enzymes have recently been proposed to increase soluble expression of foreign proteins in Escherichia coli. To date, these methods have only been used to screen for proteins with better folding properties but have never been used to select from a large library of mutants. In this paper we apply one of these methods to select mutations that increase the soluble expression of two antibody fragments in the cytoplasm of E. coli. RESULTS: We used the beta-galactosidase alpha-complementation system to monitor and evolve two antibody fragments for high expression levels in E. coli cytoplasm. After four rounds of mutagenesis and selection from large library repertoires (>107 clones), clones exhibiting high levels of beta-galactosidase activity were isolated. These clones expressed a higher amount of soluble fusion protein than the wild type in the cytoplasm, particularly in a strain deficient in the cytoplasmic Lon protease. The increase in the soluble expression level of the unfused scFv was, however, much less pronounced, and the unfused proteins proved to be more aggregation prone than the wild type. In addition, the soluble expression levels were not correlated with the beta-galactosidase activity present in the cells. CONCLUSION: This is the first report of a selection for soluble protein expression using a fusion reporter method. Contrary to anticipated results, high enzymatic activity did not correlate with the soluble protein expression level. This was presumably due to free alpha-peptide released from the protein fusion by the host proteases. This means that the alpha-complementation assay does not sense the fusion expression level, as hypothesized, but rather the amount of free released alpha-peptide. Thus, the system does not select, in our case, for higher soluble protein expression level but rather for higher protease susceptibility of the fusion protein.

Dataset Information

Expanding the utility of beta-galactosidase complementation: piece by piece.

Publications

Expanding the utility of beta-galactosidase complementation: piece by piece.

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