Development of a molecularly evolved, highly sensitive CaMKII FRET sensor with improved expression pattern.

ABSTRACT: Genetically encoded fluorescence resonance energy transfer (FRET) biosensors have been successfully used to visualize protein activity in living cells. The sensitivity and accuracy of FRET measurements directly depend on biosensor folding efficiency, expression pattern, sensitivity, and dynamic range. Here, to improve the folding efficiency of the Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) FRET biosensor, we amplified the association domain of the CaMKIIα gene using error-prone polymerase chain reaction (PCR) and fused it to the N-terminus of mCherry in a bacterial expression vector. We also created an Escherichia coli expression library based on a previously reported fluorescent protein folding reporter method, and found a bright red fluorescent colony that contained the

SUBMITTER: Shibata AC 

PROVIDER: S-EPMC4370617 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature