Cryo-EM snapshots of mycobacterial arabinosyltransferase complex EmbB-AcpM

Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) to its arabinosyl acceptor is an essential enzyme for Mtb cell wall synthesis. Analysis of drug resistance mutations suggests that EmbB is the main target of the front-line anti-TB drug, ethambutol. Herein, we report the cryo-EM structures of Mycobacterium smegmatis EmbB in its "resting state" and DPA-bound "active state". EmbB is a fifteen-transmembrane-spanning protein, assembled as a dimer. Each protomer has an associated acyl-carrier-protein (AcpM) on their cytoplasmic surface. Conformational changes upon DPA binding indicate an asymmetric movement within the EmbB dimer during catalysis. Functional studies have identified critical residues in substrate recognition and catalysis, and demonstrated that ethambutol inhibits transferase activity of EmbB by competing with DPA. The structures represent the first step directed towards a rational approach for anti-TB drug discovery.

Cryo-EM snapshots of mycobacterial arabinosyltransferase complex EmbB-AcpM

Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) to its arabinosyl acceptor is an essential enzyme for Mtb cell wall synthesis. Analysis of drug resistance mutations suggests that EmbB is the main target of the front-line anti-TB drug, ethambutol. Herein, we report the cryo-EM structures of Mycobacterium smegmatis EmbB in its "resting state" and DPA-bound "active state". EmbB is a fifteen-transmembrane-spanning protein, assembled as a dimer. Each protomer has an associated acyl-carrier-protein (AcpM) on their cytoplasmic surface. Conformational changes upon DPA binding indicate an asymmetric movement within the EmbB dimer during catalysis. Functional studies have identified critical residues in substrate recognition and catalysis, and demonstrated that ethambutol inhibits transferase activity of EmbB by competing with DPA. The structures represent the first step directed towards a rational approach for anti-TB drug discovery.

Construction of engineered exosomes with high loading efficiency of cellular endogenous proteins / 生物工程学报

Exosomes have many advantages as natural drug delivery carriers, but their application is limited by the inefficient loading of intracellular drugs (such as proteins and nucleic acids). In this study, mCherry, a red fluorescent protein, was used as the endogenous cargo target. Through gene modification of donor cells and fusion expression of membrane localization elements (PB, CAAX, Palm and CD63), mCherry was specifically sorted into exosomes through biogenesis. Results show that CD63 had the highest sorting efficiency, followed by Palm. PB and CAAX led enrichment of mCherry on the plasma membrane, but not in exosomes. The approach provides an alternative to facilitate packaging of cargo by exosomes and thus to increase the efficient delivery of endogenous protein drugs.

Preface for special issue on synthetic biology (2017) / 生物工程学报

Synthetic biology has developed quickly over the past decade. To review the research progress in synthetic biology, we published this special issue that consists of three columns, namely scientific significance, technological advances, and applications in medical science, pharmaceutics, agriculture, material, environment and energy.

Synthesis of multi-dimensional nano biostructures and biodevices through self-assembly of biomacromolecules / 生物工程学报

There are various nanostructures in biological system. They are made of biological molecules via self-assembly with well-organized architectures and specific functions. These nanostructures can be grouped into lines, layers and cages, corresponding to one-, two- and three-dimensional structures, respectively. The bionanostructures can serve as the models or templates for biosynthesis of biodevices with desired functions by rational design. Proof-of-concept studies have shown their attractive performance in practical applications, e.g., biosensors, catalysis, tumor thermo-therapy, drug delivery, tissue engineering, and batteries.

A simplified method for reconstituting active E. coli DNA polymerase III

Genome duplication in E. coli is carried out by DNA polymerase III, an enzyme complex consisting of ten subunits. Investigations of the biochemical and structural properties of DNA polymerase III require the expression and purification of subunits including α, ge, θ, γ, δ', δ, and β separately followed by in vitro reconstitution of the pol III core and clamp loader. Here we propose a new method for expressing and purifying DNA polymerase III components by utilizing a protein co-expression strategy. Our results show that the subunits of the pol III core and those of the clamp loader can be coexpressed and purified based on inherent interactions between the subunits. The resulting pol III core, clamp loader and sliding clamp can be reconstituted effectively to perform DNA polymerization. Our strategy considerably simplifies the expression and purification of DNA polymerase III and provides a feasible and convenient method for exploring other multi-subunit systems.

Development in ligase-mediated techniques for bio-molecular analysis / 生物工程学报

Two oligonucleotide probes are permitted to anneal to the nucleic acid target of interest so that the ends of two probes immediately become adjacent to each other. The ligase can then efficiently join the two juxtaposed oligonucleotide probes by the formation of a phosphodiester bond if and only if perfectly matched base-pairs at the nick are present. During past 20 years, many ligase-mediated techniques have been developed for analyzing various bio-molecules, such as known/unknown point mutations, small-scale insertions and deletions, CpG islands methylation, large sets of single nucleotide polymorphisms (SNPs), specific proteins and DNA regions with which some other proteins can interact. Since the ligation reaction can be easily integrated into other techniques, certain advances have been already achieved. These novel approaches retain high accuracy through multiple hybridization and enzymatic processing events, and provide inherent quality control checking. In this article, we provide a comprehensive review of the ligase-mediated techniques for bio-molecular analysis.

Current researches on molecular enzyme engineering / 生物工程学报

The developments of recombinant DNA technology and structural biology make it possible to modify enzyme in molecular level. Scientists show growing interests in the evolution or functional fusion of enzymes. Recent advances and applications of the molecular enzyme engineering are reviewed and discussed in this article.

High expression and identification of DNA mismatch repair gene mutS in Escherichia coli / 生物工程学报

DNA mismatch repair gene mutS (2.56 kb) was PCR modified and cloned into a secretive prokaryotic expression vector pET32a (+) which carries a N-terminal His.tag + and thioredoxin sequence. MutS protein was expressed with high level after IPTG induction using the strain E. coli AD494(DE3). SDS-PAGE revealed that the expected protein with a molecular weight of 108 kD which is about 35% of the total bacterial proteins is almost soluble. The expected protein was purified directly by immobilized metal (Ni2+) chelation affinity chromatography and the purity is over 90%. MutS protein activity verified using mismatch DNA showed that the expression product can recognize and bind to base-pair mismatch specifically.

Heterogenous Gene Expression of Methyl Parathion Hydrolase and Analysis of the Enzyme Activity / 微生物学通报

Methyl parathion hydrolase (MPH, E.C.3.1.8.1) coding gene mph from Pseudomonas sp. WBC-3, isolated and identified by our lab, was successfully expressed in E. coli AD494 (DE3)/ pET32a(+) system as soluble fusion form at high level. The recombinant MPH showed nearly 4～5 fold higher specific activity to parathion than the enzyme from Pseudomonas sp. WBC-3. In addition, the thermal stability of the recombinant enzyme was improved comparing with the wild type enzyme.