Export of recombinant proteins in Escherichia coli using ABC transporter with an attached lipase ABC transporter recognition domain (LARD)

<p>Abstract</p> <p>Background</p> <p>ATP binding cassette (ABC) transporter secretes the protein through inner and outer membranes simultaneously in gram negative bacteria. Thermostable lipase (TliA) of <it>Pseudomonas fluorescens </it>SIK W1 is secreted through the ABC transporter. TliA has four glycine-rich repeats (GGXGXD) in its C-terminus, which appear in many ABC transporter-secreted proteins. From a homology model of TliA derived from the structure of <it>P. aeruginosa </it>alkaline protease (AprA), lipase ABC transporter domains (LARDs) were designed for the secretion of fusion proteins.</p> <p>Results</p> <p>The LARDs included four glycine-rich repeats comprising a β-roll structure, and were added to the C-terminus of test proteins. Either Pro-Gly linker or Factor Xa site was added between fusion proteins and LARDs. We attached different length of LARDs such as LARD0, LARD1 or whole TliA (the longest LARD) to three types of proteins; green fluorescent protein (GFP), epidermal growth factor (EGF) and cytoplasmic transduction peptide (CTP). These fusion proteins were expressed in <it>Escherichia coli </it>together with ABC transporter of either <it>P. fluorescens </it>or <it>Erwinia chrysanthemi</it>. Export of fusion proteins with the whole TliA through the ABC transporter was evident on the basis of lipase enzymatic activity. Upon supplementation of <it>E. coli </it>with ABC transporter, GFP-LARDs and EGF-LARDs were excreted into the culture supernatant.</p> <p>Conclusion</p> <p>The LARDs or whole TliA were attached to C-termini of model proteins and enabled the export of the model proteins such as GFP and EGF in <it>E. coli </it>supplemented with ABC transporter. These results open the possibility for the extracellular production of recombinant proteins in <it>Pseudomonas </it>using LARDs or TliA as a C-terminal signal sequence.</p

Structural basis of functions of the mitochondrial cytochrome bc1 complex

AbstractThe crystal structure of the cytochrome bc1 complex (ubiquinol-cytochrome c reductase) from bovine heart submitochondria was determined at 2.9 Å resolution. The bc1 complex in crystal exists as a closely interacting dimer, suggesting that the dimer is a functional unit. Over half of the mass of the complex, including subunits core 1 and core 2, are on the matrix side of the membrane, while most of the cytochrome b subunit is located within the membrane. There are 13 transmembrane helices in each monomer, eight of them belonging to cytochrome b. Two large cavities are made of the transmembrane helices D, C, F and H in one monomer and helices D′ and E′ from the other monomer of cytochrome b, and the transmembrane helices of c1, iron-sulfur protein (ISP), and subunits 10 and 11. These cavities provide entrances for ubiquinone or inhibitor and connect the Qi pocket of one monomer and the Qo pocket of the other monomer. Ubiquinol made at the Qi site of one monomer can proceed to the nearby Qo site of the other monomer without having to leave the bc1 complex. The soluble parts of cytochrome c1 and ISP, including their redox prosthetic groups, are located on the cytoplasmic side of the membrane. The distances between the four redox centers in the complex have been determined, and the binding sites for several electron transfer inhibitors have been located. Structural analysis of the protein/inhibitor complexes revealed that the extramembrane domain of the Rieske iron-sulfur protein may undergo substantial movement during the catalytic cycle of the complex. The Rieske protein movement and the larger than expected distance between FeS and cytochrome c1 heme suggest that electron transfer reaction between FeS and cytochrome c1 may involve movements or conformational changes in the soluble domain of iron-sulfur protein. The inhibitory function of E-β-methoxyacrylate-stilbene and myxothiazol may result from the increase of mobility in ISP, whereas the function of stigmatellin and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole may result from the immobilization of ISP