Difference between revisions of "Part:BBa K3896007"

Latest revision as of 16:17, 20 October 2021

The gene of recombinant PmrCAB (DPP4) two-component system.

We chose PmrA-PmrB two-component system as the carrier of DPP4 core binding domain. The system exists in Salmonella and most strains. Initially, it is a regulatory system sensitive to Fe (III)[1]. Its transmembrane part PmrB contains a histidine kinase (HK) for sensing specific environmental stimuli and a corresponding response regulator (RR) PmrA for mediating cell responses.

The detection target is s protein, a monomer of spinous process glycoprotein on MERS-CoV envelope, which mediates the binding to human cell dipeptidyl peptidase 4 (DPP4) [2]. By reviewing the data, we found that in the complex protein of DPP4 and S protein, the core binding domain of DPP4 is concentrated between Gly260 and Asp330, while the RBD of S protein is Gln471 to Asp580 (Fig. 1). Therefore, we intercepted the polypeptide sequence of DPP4 fragment as our core detection binding domain.

Fig.1 Binding domain of S protein and hDPP4

We replaced the Fe (III) sensitive domains Trp34 to Glu64 of the original PmrB with the core binding domain of DPP4 protein to be stimulated by the S protein of MERS-CoV. After detecting the S protein of MERS-CoV, the sensor kinase PmrB autophosphorylates the highly conserved histidine residue, and then transfers the phosphate group to the conserved aspartate residue in its homologous reaction regulator PmrA. Then phosphorylated PmrA protein combined with promoter PmrC sequence to activate the expression of reporter gene.

In order to verify the detection function of our engineering bacteria, we designed a protein characterization experiment. In the experiment, 1 mm IPTG was added to induce the expression of PmrB and PmrA, and the extracted S protein was added 2 hours after the induced expression. When the engineering bacteria received the stimulation of S protein, the transmembrane protein PmrB contained a histidine kinase (HK) for autophosphorylation, and then the phosphate group was transferred to the conserved aspartate residue in the intracellular regulator PmrA, PmrA activates the transcription of PmrC promoter and expresses the downstream reporter gene EGFP.

We transferred the plasmid into E. coli BL21(DE3) and set up the following experimental groups, which yielded results consistent with expectations.

We also set up different concentration gradients and tested the fluorescence intensity and absorbance of the engineered bacterial solution, and processed the experimental data to arrive at the optimum concentration(Fig.2).

Fig.2 Fluorescence intensity of EGFP.Ctrl:Detection bacterial.IPTG:Detection bacterial+IPTG.IPTG+S Pr:Detection bacterial+IPTG+S protein

A series of experimental results demonstrate the feasibility of the PmrCAB system for MERS-CoV detection.

Reference

[1].Shangwen, Zhang , et al. "Fabrication and Characterization of One Interpenetrating Network Hydrogel Based on Sodium Alginate and Polyvinyl Alcohol." Journal of Wuhan University of Technology-Mater. Sci. Ed. 34.003(2019):744-751.

[2]Wang N, Shi X, Jiang L, Zhang S, Wang D, Tong P, Guo D, Fu L, Cui Y, Liu X, Arledge KC, Chen YH, Zhang L, Wang X. Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4. Cell Res. 2013 Aug;23(8):986-93.

Sequence and Features