Study On Bacterial Mechanosensitive Ion Channel MscL

Mechanosensitive channels（MS）,as a transmembrane channel protein,are involved in many physiological and pathological activities in organisms,such as hearing,touch,etc and have the ability to regulate in many systems,such as bones and teeth.When the extracellular osmotic pressure drops,the mechanosensitive channel of large conductance（MscL）can sense the swelling pressure,open the"hydrophobic gate",release the cell contents,maintain the osmotic pressure balance,and avoid cell lysis and death.The structure of MscL protein is simple,and it is easy to modify residues or add special response groups,so that MscL no longer only responds to changes under osmotic pressure,but to specific external stimuli（including light,ultrasound,p H,magnetic field,etc.）.Magnetic receptor protein（MagR）has been confirmed to respond to external magnetic fields in bacteria,regulating bacterial magnetic gene expression and making corresponding behaviors.At present,with the wide application of the remodeled nanoliposome system as a means of tansporting small molecule drugs or triggering tumor cell apoptosis for cancer treatment and the increasingly serious problem of bacterial antibiotic resistance,it is necessary to select appropriate channel proteins for remodeling and appropriate external stimuli to regulate the switch on and off of channel.The MscL channel can be reconstituted and active in liposomes,but we cannot regulate the unmodified MscL channel.So we choose to construct the MscL/MagR recombinant plasmid,hoping to construct a recombinant protein that has biological functions and can respond to external magnetic field.The characterization of the functional regulation of MscL protein under magnetic field will provide new research content for the research of MscL protein in nanoliposome drug delivery and bacterial antibiotic resistance,which has important research value.The research contents of this paper are as follows:1)Construction of recombinant plasmids:We constructed p ET28a[MscL]and recombinant plasmids p ET28a[MscL/MagR]by molecular cloning technology;2)Optimization of induction expression conditions:We optimized the induction temperature,inducer concentration and the induction time to obtain the maximum expression.we found that the optimal induction conditions for MscL protein were 37°C and 1 mmol/L IPTG for overnight and the optimal induction conditions for MscL/MagR proteins were 37°C and 0.5 mmol/L IPTG for 3 hours;3)Purification of protein:We confirmed the correct expression of MscL and MscL/MagR proteins on the membrane after crushing-centrifugation-ultracentrifugation.According to the expression of the target protein in the supernatant and pellet after crushing and centrifugation,it was determined that the MscL protein is suitable for non-denaturing purification,MscL/MagR recombinant protein is suitable for inclusion body purification,and we found that 5 L bacterial solution can obtain about 0.45 mg of purified MscL and 0.55 mg of purified MscL/MagR protein,and the purity is high;4)Characterization of secondary structure:We found that theα-helix structure of the two purified proteins was complete,and the secondary structure of the two purified proteins was preliminarily judged to be complete by using Circular Dichroism and Fourier Transform Infrared Spectroscopy;5)Characterization of functional activity of proteins.The functional activities of MscL and MscL/MagR recombinant proteins were characterized by Fluorescence Spectrophotometer and Isothermal Titration Calorimetry（ITC）at each level.We found that the permeability of the MscL channel to the four ions studied was K⁺>Na⁺>Co²⁺>Mn²⁺,and the binding force was Co²⁺>Mn²⁺,which enriched the ions transported by the MscL channel;at the same time,we determined that the recombinant protein MscL/MagR still has the function of gating in response to changes under osmotic pressure.It can pass K⁺,Na⁺,and Mn²⁺smoothly,except Co²⁺.It can combine with Co²⁺ions,but it has no binding heat with Mn²⁺.We think that some conformational changes during the folding process may alter the binding sites of Co²⁺and Mn²⁺.