## 4 Discussion  
Many pathogens, including _E. coli_, cannot be killed by conventional antibiotics (including colistin, a last-resort drug) due to the emergence of antibiotic resistance, which urges the development of novel antibacterial drugs [1; 2; 3]. Bacterial FtsZ protein plays an important role in cell division and may be used as one of the targets for antibacterial agents [34]. Although recent studies have revealed some natural products that may inhibit FtsZ GTPase activity and kill bacteria, there are limited known natural products used to fight _E. coli_[35]. In this work, the recombinant FtsZ protein was expressed in _E. coli_ and  
Figure 4: Interaction of berberine hydrochloride and the recombinant FtsZ protein. (**A**) Effects of different concentrations of GTP on FtsZ aggregation. Lane 1: 0 mM GTP; lane 2: 0.25 mM GTP; lane 3: 0.5 mM GTP; lane 4: 1 mM GTP; lane 5: 2 mM GTP; lane 6: 4 mM GTP; lane 7: 8 mM GTP; M: marker. Lane numbers indicate the relative intensity of the bands. (**B**) Effect of berberine hydrochloride on FtsZ aggregation. Lane 1-3: berberine hydrochloride was first incubated for 30 min with FtsZ protein and then incubated for another 30 min with GTP; lane 4-6: berberine hydrochloride was incubated directly with FtsZ protein and GTP for 1 h; berberine hydrochloride concentrations were 1.56 (lane 1 and 4), 6.25 (lane 2 and 5), and 25 (lane 3 and 6) \(\mathrm{\SIUnitSymbolMicro}\mathrm{\  
purified by the Ni\({}^{2+}\)-NTA column; a rapid and efficient screening method of antibacterial agents was mainly constructed based on the GTPase activity of FtsZ protein. The _E. coli_ expression system has been a preferred microbial cell factory for the production of many recombinant proteins (such as FtsZ, thGH, TusA, etc.) due to its advantages, such as simple procedure, low cost, scale, or high quantities [36; 37; 38]. Heterologous proteins produced in _E. coli_ usually result in soluble and insoluble proteins, also known as inclusion bodies. Expression form of heterologous proteins in _E. coli_ is related to culture and induction temperature [39]. A temperature of 37 \({}^{\circ}\)C or higher during cultivation, which causes a larger growth rate of bacteria and cell density, is more prone to form inclusion bodies, which can accumulate near the cytoplasm (or inner membrane) and block protein translocation across channels in _E. coli_[40]. In contrast, proteins expressed in _E. coli_ at lower temperatures in the range of 18 \({}^{\circ}\)C to 25 \({}^{\circ}\)C can form aggregates in the inner membrane or periplasmic space and are released to the supernatant in soluble formation; several advantages of low temperature include reduced proteolytic activity, simpler purification protocols due to fewer host proteins, correct formation of disulfide bonds, etc. [41]. Additionally, lower temperatures can stabilize refolded enzymes and lead to lower protein expression rates, so that newly transcribed recombinant proteins have enough time to fold correctly or properly [42]. As a consequence, low temperature can enhance the refolded yield of recombinant proteins produced in _E. coli_ and is therefore usually selected to induce the production of heterogenous proteins [43; 44; 45; 46]. However, some previous studies have demonstrated that lower temperatures may limit bacterial cell growth by disrupting an early step in protein synthesis, which leads to low recombinant protein yields [47; 48]. In our study, FtsZ protein was produced as an inclusion form after induction with IPTG at 16-37 \({}^{\circ}\)C (Figure 1C); the highest expression level (approximately 1 mg/mL) of FtsZ protein was obtained after induction with 0.2 mM IPTG for 5 h at 37 \({}^{\circ}\)C (Figure 1D). Furthermore, ultrasonic lysis of bacterial cells at 97.5 W was more effective than at 162.5 W or 227.5 W (Figure 2A-C), which may be linked to an increase in protein denaturation [49]. Proper concentrations of imidazole may result in a higher purity of the eluted sample.