Single protein production (SPP) system in Escherichia coli

Suzuki, Motoo; Mao, Lili; Inouye, Masayori

doi:10.1038/nprot.2007.252

Cited by 62 publications

(92 citation statements)

References 20 publications

(32 reference statements)

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“…Dedicated preparation methods (see, e.g., ref. 6) including the single-protein production method (26) are available to further reduce unwanted background contributions. Cellular ssNMR spectroscopy as described herein opens opportunities for the structural investigation of large and/or membrane-associated macromolecules.…”

Section: Discussionmentioning

confidence: 99%

Cellular solid-state nuclear magnetic resonance spectroscopy

Renault¹,

Boxtel²,

Bos³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

178

220

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Decrypting the structure, function, and molecular interactions of complex molecular machines in their cellular context and at atomic resolution is of prime importance for understanding fundamental physiological processes. Nuclear magnetic resonance is a wellestablished imaging method that can visualize cellular entities at the micrometer scale and can be used to obtain 3D atomic structures under in vitro conditions. Here, we introduce a solid-state NMR approach that provides atomic level insights into cell-associated molecular components. By combining dedicated protein production and labeling schemes with tailored solid-state NMR pulse methods, we obtained structural information of a recombinant integral membrane protein and the major endogenous molecular components in a bacterial environment. Our approach permits studying entire cellular compartments as well as cell-associated proteins at the same time and at atomic resolution. cellular envelope | Escherichia coli | lipoprotein | PagL | magic angle spinning P hysiological processes rely on the concerted action of molecular entities in and across different cellular compartments. Whereas advancements in molecular imaging have provided unprecedented insights into the macromolecular organization in the subnanometer range (1), studying atomic structure and motion in situ has been challenging for structural biology. NMR has provided insight into cellular processes (2-4) and can determine entire 3D molecular structures inside living cells (5) provided that molecular entities tumble rapidly in a cellular setting. In principle, solid-state NMR (ssNMR) spectroscopy offers a complementary spectroscopic tool to monitor molecular structure and dynamics at atomic resolution in a complex setting (see ref. 6 for a recent review). Indeed, ssNMR has already been used to study individual molecular components in the context of natural bilayers (7,8), bacterial cell walls (9), and cellular organelles (10).Here, we introduce a general approach to investigate structure and dynamics of an arbitrary molecular target and its potential molecular partners in a cellular setting. Our studies focuses on the Gram-negative bacterial cell that is characterized by a molecularly complex but architecturally unique envelope, consisting of two lipid bilayers, the inner and outer membrane (IM, OM), separated by the periplasm containing the peptidoglycan (PG) layer (Fig. 1A). The IM is a phospholipid bilayer and harbors α-helical proteins, whereas the OM is asymmetrical and consists of phospholipids, lipopolysaccharides (LPS), lipoproteins, and β-barrelfold integral membrane proteins. LPS forms the outermost layer of the OM and protects the cell against harmful compounds from the environment. PG is a large macromolecule that gives the cell its shape and rigidity.Using uniformly 13 C, 15 N-labeled cellular preparations of Escherichia coli, we characterized the structure and dynamics of a recombinant integral membrane protein (PagL) and other major endogenous molecular components of the cell envelope in...

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Section: Discussionmentioning

confidence: 99%

Cellular solid-state nuclear magnetic resonance spectroscopy

Renault¹,

Boxtel²,

Bos³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

178

220

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“…Expression constructs were transformed into pMazF-competent cells using 25 μg/mL chloramphenicol and 100 μg/mL ampicillin. Protein expression was carried out according to the SPP protocol (22).…”

Section: Methodsmentioning

confidence: 99%

“…Based on previous work (5), a Yop1p construct lacking 23 N-terminal and 15 C-terminal residues (residues 24-165; hereafter referred to as Yop1p) was chosen for structural studies. This construct was expressed in Escherichia coli using the singleprotein production (SPP) system (22), and subsequently purified and refolded into 2% (wt/vol) (42 mM) 1-myristoyl-2-hydroxy-snglycero-3-phospho-(1′-rac-glycerol) (LMPG) micelles. The similarity of the bacterially expressed Yop1p protein to the protein expressed in yeast was initially assessed by oligomerization, which is correlated with Yop1p function (3,5).…”

Section: Bacterially Expressed Yop1p Oligomerizes and Forms Membranementioning

confidence: 99%

A conserved amphipathic helix is required for membrane tubule formation by Yop1p

Brady

Claridge

Smith

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The integral membrane proteins of the DP1 (deleted in polyposis) and reticulon families are responsible for maintaining the high membrane curvature required for both smooth endoplasmic reticulum (ER) tubules and the edges of ER sheets, and mutations in these proteins lead to motor neuron diseases, such as hereditary spastic paraplegia. Reticulon/DP1 proteins contain reticulon homology domains (RHDs) that have unusually long hydrophobic segments and are proposed to adopt intramembrane helical hairpins that stabilize membrane curvature. We have characterized the secondary structure and dynamics of the DP1 family protein produced from the YOP1 gene (Yop1p) and identified a C-terminal conserved amphipathic helix (APH) that, on its own, interacts strongly with negatively charged membranes and is necessary for membrane tubule formation. Analyses of DP1 and reticulon family members indicate that most, if not all, contain C-terminal sequences capable of forming APHs. Together, these results indicate that APHs play a previously unrecognized role in RHD membrane curvature stabilization.T he endoplasmic reticulum (ER) is the largest membranebound organelle in the eukaryotic cell and adopts diverse morphologies, including tubules that extend outward to the cell periphery and sheet-like structures closer to the nucleus (1, 2). The regions of high membrane curvature that are found in the ER tubules and sheets are stabilized by the DP1 (deleted in polyposis) and reticulon classes of integral membrane proteins (3-5). Consistent with their role in ER morphology, similar proteins have not been found in prokaryotes (6).There are six human DP1 proteins originally identified as accessory proteins facilitating the expression of odorant receptors, and thus termed receptor expression-enhancing proteins (REEPs) (7). The ability of REEPs to facilitate receptor trafficking has recently been related back to their ER shaping activities (8). The importance of REEPs in ER morphology is also highlighted by their implication in human diseases that are associated with neurons having long axons and requiring an extended tubular ER (9, 10). For example, mutations in the transmembrane domain of REEP1, which is primarily expressed in neurons (11), lead to pure forms of hereditary spastic paraplegia (HSP) (12, 13).The reticulon family in humans comprises four members, Rtn1-Rtn4 (6). Rtn4 is of particular interest because it is primarily responsible for generating or stabilizing the tubular ER in mammalian cells. Rtn4 is critical in neuronal cell processes because it inhibits spontaneous neurite outgrowth (14) and restricts neuronal plasticity (15), and it has been implicated in several neural diseases, including schizophrenia and motor neuron disease (16-18).The DP1 and reticulon proteins have in common a region containing two, unusually long hydrophobic segments (≈35 aa in length each) known as a reticulon homology domain (RHD) (6). In humans, REEPs 1-6 are similar overall; however, the RHD of REEPs 1-4 is truncated (Fig. S1). REEPs 1-4 also possess...

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“…This system is thus termed the single-protein production (SPP) system. One of the most remarkable advantages of the SPP system is that the cell culture can be highly condensed without affecting protein yields (7,12,13). Using this cSPP system, one can achieve a cost savings of as much as 97.5% by condensing a culture 40-fold.…”

mentioning

confidence: 95%

Use of Amino Acids as Inducers for High-Level Protein Expression in the Single-Protein Production System

Vaiphei

Mao

Shimazu

et al. 2010

Appl Environ Microbiol

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By taking advantage of MazF, an ACA codon-specific mRNA interferase, Escherichia coli cells can be converted into a bioreactor producing only a single protein of interest by using an ACA-less mRNA for the protein. In this single-protein production (SPP) system, we engineered MazF by replacing two tryptophan residues in positions 14 and 83 with Phe (W14F) and Leu (W83L), respectively. Upon the addition of an inducer (IPTG [isopropyl-␤-D-thiogalactopyranoside]), the mutated MazF [MazF(⌬W)] can still be produced even in the absence of tryptophan in the medium by using a Trp auxotroph, while a target protein having Trp residues cannot be produced. However, at 3 h after the addition of IPTG, the addition of tryptophan to the medium exclusively induces production of the target protein at a high level. A similar SPP system was also constructed with the use of a His-less protein [MazF(⌬H)] and a His auxotroph. Using these dual-induction systems, isotopic enrichments of 13 C, 15 N, and 2 H were highly improved by almost complete suppression of the production of the unlabeled target protein. In both systems, isotopic incorporation reached more than 98% labeling efficiency, significantly reducing the background attributable to the unlabeled target protein.

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Single protein production (SPP) system in Escherichia coli

Cited by 62 publications

References 20 publications

Cellular solid-state nuclear magnetic resonance spectroscopy

Cellular solid-state nuclear magnetic resonance spectroscopy

A conserved amphipathic helix is required for membrane tubule formation by Yop1p

Use of Amino Acids as Inducers for High-Level Protein Expression in the Single-Protein Production System

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