Characterization of a novel method for the production of single-span membrane proteins in Escherichia coli.

The large-scale production and isolation of recombinant protein is a central element of the biotechnology industry and many of the products have proved extremely beneficial for therapeutic medicine. Escherichia coli is the microorganism of choice for the expression of heterologous proteins for therapeutic application, and a range of high-value proteins have been targeted to the periplasm using the well characterized Sec protein export pathway. More recently, the ability of the second mainstream protein export system, the twin-arginine translocase, to transport fully-folded proteins into the periplasm of not only E. coli, but also other Gram-negative bacteria, has captured the interest of the biotechnology industry. In this study, we have used a novel approach to block the export of a heterologous Tat substrate in the later stages of the export process, and thereby generate a single-span membrane protein with the soluble domain positioned on the periplasmic side of the inner membrane. Biochemical and immuno-electron microscopy approaches were used to investigate the export of human growth hormone by the twin-arginine translocase, and the generation of a single-span membrane-embedded variant. This is the first time that a bonafide biotechnologically relevant protein has been exported by this machinery and visualized directly in this manner. The data presented here demonstrate a novel method for the production of single-span membrane proteins in E. coli.

Probing the quality control mechanism of the Escherichia coli twin-arginine translocase with folding variants of a de novo-designed heme protein.

Protein transport across the cytoplasmic membrane of bacterial cells is mediated by either the general secretion (Sec) system or the twin-arginine translocase (Tat). The Tat machinery exports folded and cofactor-containing proteins from the cytoplasm to the periplasm by using the transmembrane proton motive force as a source of energy. The Tat apparatus apparently senses the folded state of its protein substrates, a quality-control mechanism that prevents premature export of nascent unfolded or misfolded polypeptides, but its mechanistic basis has not yet been determined. Here, we investigated the innate ability of the model Escherichia coli Tat system to recognize and translocate de novo-designed protein substrates with experimentally determined differences in the extent of folding. Water-soluble, four-helix bundle maquette proteins were engineered to bind two, one, or no heme b cofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional 1H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of the E. coli trimethylamine-N-oxide (TMAO) reductase (TorA) to the N terminus of the protein maquettes was sufficient for the Tat system to recognize them as substrates. The clear correlation between the level of Tat-dependent export and the degree of heme b-induced folding of the maquette protein suggested that the membrane-bound Tat machinery can sense the extent of folding and conformational flexibility of its substrates. We propose that these artificial proteins are ideal substrates for future investigations of the Tat system's quality-control mechanism.

The Prevalence and Treatment of Hip Dysplasia in Prader-Willi Syndrome (PWS).

BACKGROUND: Prader-Willi syndrome (PWS) is a genetic disorder with multisystem involvement. There are a number of associated orthopaedic manifestations, the most recognized of which is scoliosis. The aim of this study was to assess the prevalence of hip dysplasia and to investigate its treatment in patients with PWS. METHODS: Following IRB approval, all patients seen at our institution's Prader-Willi multidisciplinary clinic were retrospectively reviewed. Only patients with an ultrasound, anteroposterior (AP) spine, AP abdomen, AP hip radiograph, and/or skeletal survey were included in the study. The presence of hip dysplasia was determined based on ultrasonographic and/or radiographic measurements performed by a single fellowship trained pediatric orthopaedic surgeon. A multivariable logistic regression analysis was used to test the association between patient demographics and the prevalence of hip dysplasia. Age at diagnosis, treatment type, and outcomes were recorded for patients that underwent treatment for hip dysplasia. RESULTS: Hip dysplasia was identified in 30% (27/90) of the patient population. Two of the 27 patients (7.4%) had normal films but had a history of resolved hip dysplasia. Prevalence was not associated with sex (P=0.7072), genetic subtype (P=0.5504), race (P=0.8537), ethnicity (P=0.2191), or duration of follow-up (P=0.4421). Eight of the 27 patients (30%) underwent hip treatment by Pavlik harness (2/8), Pavlik harness and closed reduction (1/8), closed reduction (3/8), open reduction (1/8), and unspecified hip surgery (1/8). The mean age at diagnosis was 2 months for the patients that were successfully treated for hip dysplasia (3/8) and 12 months for those who had residual dysplasia following the treatment (5/8). CONCLUSIONS: Our study demonstrates a higher prevalence of hip dysplasia in patients with PWS than previously documented. The age at which hip dysplasia develops remains unknown; therefore, we recommend an ultrasound screening for all infants with PWS at 6 weeks of age and subsequent radiographic studies at 1, 2, 5, 10, and 15 years of age to allow for early diagnosis and intervention. LEVEL OF EVIDENCE: Level III-retrospective comparative study.

The Bacillus subtilis TatAdCd system exhibits an extreme level of substrate selectivity.

The Tat system preferentially transports correctly folded proteins across the bacterial membrane although little is known of the proofreading mechanism. Most research has focused on TatABC systems from Gram-negative bacteria, especially Escherichia coli, and much less is known of the TatAC-type systems from Gram-positive organisms. We have previously shown that the Bacillus subtilis TatAdCd system is functional in an E. coli tat null background and able to transport TorA-GFP and native TorA (TMAO reductase); here, we examined its ability to transport other proteins bearing a TorA signal sequence. We show that whereas E. coli TatABC transports a wide range of biotherapeutics including human growth hormone, interferon α2b, a VH domain protein and 2 different scFvs, TatAdCd transports the scFvs but completely rejects the other proteins. The system also rejects two native E. coli substrates, NrfC and FhuD. Moreover, we have shown that TatABC will transport a wide range of folded scFv variants with the surface altered to incorporate multiple salt bridges, charged residues (5 glutamate, lysine or arginine), or hydrophobic residues (up to 6 leucines). In contrast, TatAdCd completely rejects many of these variants including those with 5 or 6 added Leu residues. The combined data show that the TatABC and TatAdCd systems have very different substrate selectivities, with the TatAdCd system displaying an extreme level of selectivity when compared to the E. coli system. The data also provide a preliminary suggestion that TatAdCd may not tolerate substrates that contain surface domains with a level of hydrophobicity above a certain threshold.

Proofreading of substrate structure by the Twin-Arginine Translocase is highly dependent on substrate conformational flexibility but surprisingly tolerant of surface charge and hydrophobicity changes.

The Tat system transports folded proteins across the bacterial plasma membrane, and in Escherichia coli preferentially transports correctly-folded proteins. Little is known of the mechanism by which Tat proofreads a substrate's conformational state, and in this study we have addressed this question using a heterologous single-chain variable fragment (scFv) with a defined structure. We introduced mutations to surface residues while leaving the folded structure intact, and also tested the importance of conformational flexibility. We show that while the scFv is stably folded and active in the reduced form, formation of the 2 intra-domain disulphide bonds enhances Tat-dependent export 10-fold, indicating Tat senses the conformational flexibility and preferentially exports the more rigid structure. We further show that a 26-residue unstructured tail at the C-terminus blocks export, suggesting that even this short sequence can be sensed by the proofreading system. In contrast, the Tat system can tolerate significant changes in charge or hydrophobicity on the scFv surface; substitution of uncharged residues by up to 3 Lys-Glu pairs has little effect, as has the introduction of up to 5 Lys or Glu residues in a confined domain, or the introduction of a patch of 4 to 6 Leu residues in a hydrophilic region. We propose that the proofreading system has evolved to sense conformational flexibility and detect even very transiently-exposed internal regions, or the presence of unfolded peptide sections. In contrast, it tolerates major changes in surface charge or hydrophobicity.