Active Transport of Membrane Components by Self-Organization of the Min Proteins.

Heterogeneous distribution of components in the biological membrane is critical in the process of cell polarization. However, little is known about the mechanisms that can generate and maintain the heterogeneous distribution of the membrane components. Here, we report that the propagating wave patterns of the bacterial Min proteins can impose steric pressure on the membrane, resulting in transport and directional accumulation of the component in the membrane.

The C-terminal D/E-rich domain of MBD3 is a putative Z-DNA mimic that competes for Zα DNA-binding activity.

The Z-DNA binding domain (Zα), derived from the human RNA editing enzyme ADAR1, can induce and stabilize the Z-DNA conformation. However, the biological function of Zα/Z-DNA remains elusive. Herein, we sought to identify proteins associated with Zα to gain insight into the functional network of Zα/Z-DNA.

Crystal structure of vespid phospholipase A(1) reveals insights into the mechanism for cause of membrane dysfunction.

Vespid phospholipase A1 (vPLA1) from the black-bellied hornet (Vespa basalis) catalyzes the hydrolysis of emulsified phospholipids and shows potent hemolytic activity that is responsible for its lethal effect. To investigate the mechanism of vPLA1 towards its function such as hemolysis and emulsification, we isolated vPLA1 from V. basalis venom and determined its crystal structure at 2.

Linked production of pyroglutamate-modified proteins via self-cleavage of fusion tags with TEV protease and autonomous N-terminal cyclization with glutaminyl cyclase in vivo.

Overproduction of N-terminal pyroglutamate (pGlu)-modified proteins utilizing Escherichia coli or eukaryotic cells is a challenging work owing to the fact that the recombinant proteins need to be recovered by proteolytic removal of fusion tags to expose the N-terminal glutaminyl or glutamyl residue, which is then converted into pGlu catalyzed by the enzyme glutaminyl cyclase. Herein we describe a new method for production of N-terminal pGlu-containing proteins in vivo via intracellular self-cleavage of fusion tags by tobacco etch virus (TEV) protease and then immediate N-terminal cyclization of passenger target proteins by a bacterial glutaminyl cyclase. To combine with the sticky-end PCR cloning strategy, this design allows the gene of target proteins to be efficiently inserted into the expression vector using two unique cloning sites (i.

Learning to predict expression efficacy of vectors in recombinant protein production.

Background: Recombinant protein production is a useful biotechnology to produce a large quantity of highly soluble proteins. Currently, the most widely used production system is to fuse a target protein into different vectors in Escherichia coli (E. coli).

Engineering a novel endopeptidase based on SARS 3CL(pro).

A 3C-like protease (3CLpro) from the severe acute respiratory syndrome-coronavirus (SARS-CoV) is required for viral replication, cleaving the replicase polyproteins at 11 sites with the conserved Gln [downward arrow](Ser, Ala, Gly) sequences. In this study, we developed a mutant 3CLpro (T25G) with an expanded S1' space that demonstrates 43.5-fold better k(cat)/K(m) compared with wild-type in cleaving substrates with a larger Met at P1' and is suitable for tag removal from recombinant fusion proteins.

Parallel gene cloning and protein production in multiple expression systems.

To quickly find an optimal expression system for recombinant protein production, a set of vectors with the same restriction sites were constructed for parallel cloning of a target gene and recombinant protein production in prokaryotic and eukaryotic expression systems, simultaneously. These vectors include nucleotide sequences encoding protein tags and protease recognition sites for tag removal, followed by the cloning sites 5'-EcoRI/3'-XhoI identical in these vectors for ligating with the sticky-end PCR product of a target gene. Our vectors allow parallel gene cloning and protein production in multiple expression systems with minimal cloning effort.

Self-cleavage of fusion protein in vivo using TEV protease to yield native protein.

Overproduction of proteins from cloned genes using fusion protein expression vectors in Escherichia coli and eukaryotic cells has increased the quantity of protein produced. This approach has been widely used in producing soluble recombinant proteins for structural and functional analysis. One major disadvantage, however, of applying this approach for clinical or bioindustrial uses is that proteolytic removal of the fusion carrier is tedious, expensive, and often results in products with additional amino acid residues than the native proteins.

High-throughput screening of soluble recombinant proteins.

The aims of high-throughput (HTP) protein production systems are to obtain well-expressed and highly soluble proteins, which are preferred candidates for use in structure-function studies. Here, we describe the development of an efficient and inexpensive method for parallel cloning, induction, and cell lysis to produce multiple fusion proteins in Escherichia coli using a 96-well format. Molecular cloning procedures, used in this HTP system, require no restriction digestion of the PCR products.