Cell-free protein expression system is a new method to express target protein in vitro and has been widely applied to the study of protein structure, protein function and other related fields. Preparation of cell extract is one of the key factors that affect the efficiency of the cell-free system. To improve the efficiency and economical feasibility of cell-free protein synthesis, we discussed the parameters during the preparation of the cell extract. These parameters include centrifugation speed, pre-incubation, and dialysis. We used the green fluorescent protein as the reporter protein, and obtained a simple procedure for the preparation of Escherichia coli cell extract. A simple centrifugation step (12 000 x g, 10 min) followed by a brief incubation was sufficient for the preparation of an active cell extract to support protein expression with higher productivity (209 microg/mL). Compared to the traditional E. coli S30 procedure, the processing time was reduced by 62%, and the productivity was increased by 2.6 times. The new procedure will make the advantage of cell-free technology more obvious, and promote its wider application.
Cell Fractionation ; methods ; Cell-Free System ; Escherichia coli ; cytology ; genetics ; metabolism ; Escherichia coli Proteins ; biosynthesis ; chemistry ; isolation & purification ; Green Fluorescent Proteins ; metabolism

Cyclic lipopeptide has extensive application prospect in the field of medicine due to its unique chemical structure and biological activity. This study aims to obtain high purity of cyclic lipopeptide monomer from Bacillus amyloliquefaciems strain Q-426, and illuminate preliminary antitumor mechanism of C-15 Bacillomycin D and C-16 Bacillomycin D. Firstly, crude cyclic lipopeptide solution was prepared by two-steps purification of acid precipitation and double-resins chromatography. In order to obtain purer product preparative HPLC was utilized to separate and purify cyclic lipopeptide. Component 1 and component 2 were detected as C-15 Bacillomycin D and C-16 Bacillomycin D by HPLC-MS and ESI-MS/MS. Secondly, the effect of C-15 Bacillomycin D, C-16 Bacillomycin D and their mixture (1:1, mol:mol) on cell proliferation was measured using human cancer cells (Hela, MG, Hep-G2 and HT-29). The cyclic peptide showed a dose dependent manner on the cell proliferation inhibition of Hela and MG cells. Finally, the results of the scratch wound healing assay and FACS analysis revealed that C-16 Bacillomycin D can effectively influence the cells migration and the cells treated with C-16 Bacillomycin D showed typical apoptotic morphology with the increase of drug concentration in the early apoptosis, late apoptosis percentage increased, and G₀G₁ arrest was induced significantly.

Based on polyketide syntheses gene (PKS) and non-ribosomal peptide synthetases gene (NRPS), one strain with high anti-pathogenic activity was screened from 77 strains isolated from Arctic marine sediments and identified. By optimizing the composition of culture medium and fermentation conditions, the production of this strain's active metabolites was improved and the main metabolites were identified by HRMS, ¹H NMR and ¹³C NMR. The antibacterial spectrum of the main metabolites and the effect of the metabolites on cucumber Fusarium wilt were also determined. The results showed that the strain was Bacillus velezensis and it showed growth promoting effect on plants. When the strain was cultured in 5 g/L maltose, 10 g/L tryptone, 10 g/L sodium chloride, at 30 ℃, 150 r/min for 60 h, the diameter of the inhibition zone increased from (16.23±0.42) to (24.42±0.57) mm. The metabolites of this strain mainly contain macrolide compound macrolactin A, which has antagonistic effect on a variety of pathogenic bacteria and fungi. Cucumber seedling experiments showed that the metabolites of this strain had a protective effect on cucumber Fusarium wilt, and showed a good potential for development and application as a biocontrol agent.
Polyketides/pharmacology* ; Fungi ; Bacteria ; Fusarium/genetics* ; Anti-Bacterial Agents/pharmacology* ; Peptide Synthases/genetics*

Fusobacterium nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues, affecting multiple stages of colorectal cancer development. The two-component system plays an important role in the regulation and expression of genes related to pathogenic resistance and pathogenicity. In this paper, we focused on the CarRS two-component system of F. nucleatum, and the histidine kinase protein CarS was recombinantly expressed and characterized. Several online software such as SMART, CCTOP and AlphaFold2 were used to predict the secondary and tertiary structure of the CarS protein. The results showed that CarS is a membrane protein with two transmembrane helices and contains 9 α-helices and 12 β-folds. CarS protein is composed of two domains, one is the N-terminal transmembrane domain (amino acids 1-170), the other is the C-terminal intracellular domain. The latter is composed of a signal receiving domain (histidine kinases, adenylyl cyclases, methyl-accepting proteins, prokaryotic signaling proteins, HAMP), a phosphate receptor domain (histidine kinase domain, HisKA), and a histidine kinase catalytic domain (histidine kinase-like ATPase catalytic domain, HATPase_c). Since the full-length CarS protein could not be expressed in host cells, a fusion expression vector pET-28a(+)-MBP-TEV-CarS_cyto was constructed based on the characteristics of secondary and tertiary structures, and overexpressed in Escherichia coli BL21-Codonplus(DE3)RIL. CarS_cyto-MBP protein was purified by affinity chromatography, ion-exchange chromatography, and gel filtration chromatography with a final concentration of 20 mg/ml. CarS_cyto-MBP protein showed both protein kinase and phosphotransferase activities, and the MBP tag had no effect on the function of CarS_cyto protein. The above results provide a basis for in-depth analysis of the biological function of the CarRS two-component system in F. nucleatum.
Humans ; Histidine Kinase/metabolism* ; Fusobacterium nucleatum/metabolism* ; Automobiles ; Protein Kinases/genetics* ; Escherichia coli/metabolism* ; Colorectal Neoplasms