Disulfide bonds formed between pairs of cysteines are important features of
the structure of many proteins. Elaborate electron transfer pathways have
evolved
Escherichia coli
to promote the formation ...of these covalent
bonds and to ensure that the correct pairs of cysteines are joined in the final
folded protein. These transfers of electrons consist, in the main, of cascades
of disulfide bond formation or reduction steps between a series of proteins
(DsbA, DsbB, DsbC, and DsbD). A surprising variety of mechanisms and protein
structures are involved in carrying out these steps.
The Gateway recombination system is characterized by its ability to transfer DNA sequences back and forth between an intermediate clone (the entry clone) and a variety of destination vectors. ...However, a number of applications do not need to exploit the advantages offered by the entry clone. Here we report reaction conditions for cloning DNA fragments into destination vectors in a single step reaction, thus reducing the cost and overall time needed to obtain an expression clone from three days to one.
The cytoplasmic membrane protein DsbD transfers electrons from the cytoplasm to the periplasm of
E. coli, where its reducing power is used to maintain cysteines in certain proteins in the reduced ...state. We split DsbD into three structural domains, each containing two essential cysteines. Remarkably, when coexpressed, these truncated proteins restore DsbD function. Utilizing this three piece system, we were able to determine a pathway of the electrons through DsbD. Our findings strongly suggest that the pathway is based on a series of multistep redox reactions that include direct interactions between thioredoxin and DsbD, and between DsbD and its periplasmic substrates. A thioredoxin-fold domain in DsbD appears to have the novel role of intramolecular electron shuttle.
G protein coupled receptors (GPCRs) represent the largest family of membrane proteins in the human genome and the richest source of targets for the pharmaceutical industry. A major limitation to ...characterizing GPCRs has been the difficulty in developing high-level heterologous expression systems that are cost effective. Reasons for these difficulties include inefficient transport and insertion in the plasma membrane and cytotoxicity. Additionally, GPCR purification requires detergents, which have a negative effect on receptor yields and stability.
Here we report a detergent-free cell-free protein expression-based method to obtain pharmacologically active GPCRs in about 2 hours. Our strategy relies on the co-translational insertion of modified GPCRs into nanometer-sized planar membranes. As a model we employed an engineered β2-adrenergic receptor in which the third intracellular loop has been replaced with T4 lysozyme (β2AR -T4L). We demonstrated that nanolipoprotein particles (NLPs) are necessary for expression of active β2AR -T4L in cell-free systems. The binding specificity of the NLP- β2AR-T4L complex has been determined by competitive assays. Our results demonstrate that β2AR-T4L synthesized in vitro depends on similar oxidative conditions as those required by an in vivo-expressed receptor.
Although the activation of β2AR-T4L requires the insertion of the T4 lysozyme sequence and the yield of that active protein limited, our results conceptually prove that cell-free protein expression could be used as a fast approach to express these valuable and notoriously difficult-to-express proteins.
The central hydrophobic domain of the membrane protein DsbD catalyzes the transfer of electrons from the cytoplasm to the periplasm of Escherichia coli. Two cysteine residues embedded in ...transmembrane segments are essential for this process. Our results, based on cysteine alkylation and site-directed proteolysis, provide strong evidence that these residues are capable of forming an intramolecular disulfide bond. Also, by using a combination of two complementary genetic approaches, we show that both cysteines appear to be solvent-exposed to the cytoplasmic side of the inner membrane. These data are inconsistent with earlier topological models that place these residues on opposite sides of the membrane and permit the formulation of alternate hypotheses for the mechanism of this unusual transmembrane electron transfer.
Membrane-associated proteins and protein complexes account for approximately a third or more of the proteins in the cell (1, 2). These complexes mediate essential cellular processes; including signal ...transduc-tion, transport, recognition, bioenergetics and cell-cell communication. In general, membrane proteins are challenging to study because of their insolubility and tendency to aggregate when removed from their protein lipid bilayer environment. This chapter is focused on describing a novel method for producing and solubilizing membrane proteins that can be easily adapted to high-throughput expression screening. This process is based on cell-free transcription and translation technology coupled with nanolipoprotein par ticles (NLPs), which are lipid bilayers confined within a ring of amphipathic protein of defined diameter. The NLPs act as a platform for inserting, solubilizing and characterizing functional membrane proteins. NLP component proteins (apolipoproteins), as well as membrane proteins can be produced by either traditional cell-based or as discussed here, cell-free expression methodologies.
Recent technical advances have revitalized cell-free expression systems to meet the increasing demands for protein synthesis. Cell-free systems offer several advantages over traditional cell-based ...expression methods, including the easy modification of reaction conditions to favor protein folding, decreased sensitivity to product toxicity and suitability for high-throughput strategies because of reduced reaction volumes and process time. Moreover, improvements in translation efficiency have resulted in yields that exceed a milligram of protein per milliliter of reaction mix. We review the advances on this expanding technology and highlight the growing list of associated applications.
Membrane protein expression: no cells required Katzen, Federico; Peterson, Todd C; Kudlicki, Wieslaw
Trends in biotechnology (Regular ed.),
08/2009, Volume:
27, Issue:
8
Journal Article
Peer reviewed
Structural and functional studies of membrane proteins have been severely hampered by difficulties in producing sufficient quantities of properly folded protein products. It is well established that ...cell-based expression of membrane proteins is generally problematic and frequently results in low yield, cell toxicity, protein aggregation and misfolding. Owing to its inherent open nature, cell-free protein expression has become a highly promising tool for the fast and efficient production of these difficult-to-express proteins. Here we review the most recent advances in this field, underscoring the potentials and weaknesses of the newly developed approaches and place specific emphasis on the use of nanolipoprotein particles (NLPs or nanodiscs).
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