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High-throughput split-protein profiling by combining transposon mutagenesis and regulated protein-protein interactions with deep sequencing
Splitting a protein at a position may lead to self- or assisted-complementary fragments depending on whether two resulting fragments can reconstitute to maintain the native function spontaneously or require assistance from two interacting molecules. Assisted complementary fragments with high contras...
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Published in: | International journal of biological macromolecules 2022-04, Vol.203, p.543-552 |
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Main Authors: | , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites |
Online Access: | Get full text |
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Summary: | Splitting a protein at a position may lead to self- or assisted-complementary fragments depending on whether two resulting fragments can reconstitute to maintain the native function spontaneously or require assistance from two interacting molecules. Assisted complementary fragments with high contrast are an important tool for probing biological interactions. However, only a small number of assisted-complementary split-variants have been identified due to manual, labour-intensive optimization of a candidate gene. Here, we introduce a technique for high-throughput split-protein profiling (HiTS) that allows fast identification of self- and assisted complementary positions by transposon mutagenesis, a rapamycin-regulated FRB-FKBP protein interaction pair, and deep sequencing. We test this technique by profiling three antibiotic-resistant genes (fosfomycin-resistant gene, fosA3, erythromycin-resistant gene, ermB, and chloramphenicol-resistant gene, catI). Self- and assisted complementary fragments discovered by the high-throughput technique were subsequently confirmed by low-throughput testing of individual split positions. Thus, the HiTS technique provides a quicker alternative for discovering the proteins with suitable self- and assisted-complementary split positions when combining with a readout such as fluorescence, bioluminescence, cell survival, gene transcription or genome editing. |
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ISSN: | 0141-8130 1879-0003 |
DOI: | 10.1016/j.ijbiomac.2022.01.173 |