DERB
Dual expression recombinase based (DERB) single vector system is a method of efficient cloning and subcloning of plasmid vectors for high throughput screening (HTS) and verification of protein-protein interactions inside living cells. DERB was developed by Lu JP et al.[1]
The DERB process
Plasmid Vectors are deliberately constructed circular double-strand DNA loops capable of self-amplification and protein production. They are widely used in laboratories and the bio-medical and pharmaceutical industries to produce of DNA or protein in quantity. The DERB vector system consists of a series of vectors, each of which produces two or more proteins which are labeled or tagged for screening and verification of molecular interactions such as protein–protein interactions.
The following characteristics of the vectors ensure highly efficient cloning or subcloning of the protein of interest ORFs into the DERB vectors for high-throughput screening (HTS) and verification of protein–protein interactions after single introducing of the vectors into eukaryotic or prokaryotic cells:
- The sequences of two or more separate sets of promoters.
- Reporter tags for FRET, BiFC, PFC etc. detection.
- Recombinase recognizable sites for the insertion of ORF of the proteins of interest.
- Selection securities for efficient cloning.
The system eliminates the requirements of multiple step restriction-purification-ligation subcloning and co-transfection, which are bottle-neck in automation in HTS protein–protein interactions.
The examination of the protein–protein interactions of interest with the DERB vector system can use standard laboratory equipment, e.g. plate reader, flow cytometer, fluorescent microscope and confocal microscope.
DERB is a new tool in high-throughput screening (HTS) and verification of protein–protein interactions and their modulators for both basic research and development such as screen new drugs or medicines for prevention and treatment of disease.