Investigation of inclusion body formation in recombinant Escherichia coli with a bioimaging system.
A bioimaging system using giant protoplasts of recombinant Escherichia coli was developed for examining conditions affecting the formation of inclusion bodies. After the giant protoplasts were prepared, isopropyl-beta-d-thiogalactopyranoside (IPTG) was used to induce expression of the blue fluorescence protein (BFP) gene. Fluorescence microscopy revealed that two types of fluorescence were emitted; one by soluble BFP and the other by aggregated BFP. As a result, the parameters that influence the formation of inclusion bodies can be examined by observing the fluorescence emitted by BFP, in real-time, with a fluorescence microscope. It was found that the aggregated BFP decreased with decreasing cultivation temperature, and the formation of inclusion bodies increased with decreased pH and increased IPTG concentrations. In addition, the aggregated form of BFP emitted fluorescence, indicating that BFP inclusion bodies might remain, at least partially, in a properly folded native-like form.
Li
Ruei-Yu
RY
Department of Chemical Engineering, National Cheng Kung University, No.1 University Road, Tainan 70101, Taiwan.
Cheng
Chu-Yuan
CY
D004926:Escherichia coli / Q000502:physiology*; D007089:Image Enhancement / Q000379:methods*; D002479:Inclusion Bodies / Q000648:ultrastructure*; D008856:Microscopy, Fluorescence / Q000379:methods*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism*
D011994:Recombinant Proteins