Positive selection improves the efficiency of DNA assembly.

With the advent of synthetic biology and cell engineering, the demand for large synthetic DNA fragments has been steadily increasing. Consequently, a number of multi-fragment cloning technologies optimized for the assembly of sizable DNA constructs have been developed. Still, screening for the right clone can be tedious because the high incidence of illegitimate assembly results in a relatively large proportion of missing or shuffled DNA elements. To mitigate this risk, we have developed a strategy that reduces the rate of fragment mis-assembly and is compatible with a variety of cloning methodologies. The approach is based on the positive selection of truncated plasmid markers, which are rendered active by providing their missing sequences during the assembly process. The method has been successfully validated in the context of complex in vivo and in vitro homologous recombination workflows, but it could be readily adapted to other cloning strategies, including those based on restriction endonucleases.

A method for multi-site-directed mutagenesis based on homologous recombination.

We have developed an efficient method for the simultaneous introduction of up to three mutations in a plasmid DNA via homologous recombination. The strategy is compatible with a variety of mutations, including degenerate codons in plasmids of different sizes. In contrast to other methodologies, this approach employs the same set of reagents for both single- and multi-site mutagenesis assays, minimizes the required protocol steps, and exhibits remarkably high mutagenesis efficiencies.

Cell-free synthesis of a functional G protein-coupled receptor complexed with nanometer scale bilayer discs.

BACKGROUND: 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. RESULTS: 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 ß2-adrenergic receptor in which the third intracellular loop has been replaced with T4 lysozyme (ß2AR -T4L). We demonstrated that nanolipoprotein particles (NLPs) are necessary for expression of active ß2AR -T4L in cell-free systems. The binding specificity of the NLP- ß2AR-T4L complex has been determined by competitive assays. Our results demonstrate that ß2AR-T4L synthesized in vitro depends on similar oxidative conditions as those required by an in vivo-expressed receptor. CONCLUSIONS: Although the activation of ß2AR-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.

Cell-free co-expression of functional membrane proteins and apolipoprotein, forming soluble nanolipoprotein particles.

Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Delta1-49 apolipoprotein A-I fragment (Delta49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR --> M transition. Importantly the functional bR was solubilized in discoidal bR.NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Delta49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies.

Cell-free expression for nanolipoprotein particles: building a high-throughput membrane protein solubility platform.

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.

Efficient generation of insect-based cell-free translation extracts active in glycosylation and signal sequence processing.

A novel method for generation of insect-based cell-free translation extracts is presented. The protocol can be completed in less than an hour, and the resulting extracts are extremely proficient in N-linked glycosylation and signal sequence processing. No specialized equipment other than that usually present in an ordinary biochemistry laboratory is required. The novel approach dramatically reduces cost and time while rendering enhanced lysates compared to previously published strategies.

The past, present and future of cell-free protein synthesis.

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.

DNA Assembly Tools and Strategies for the Generation of Plasmids.

Since the discovery of restriction enzymes and the generation of the first recombinant DNA molecule over 40 years ago, molecular biology has evolved into a multidisciplinary field that has democratized the conversion of a digitized DNA sequence stored in a computer into its biological counterpart, usually as a plasmid, stored in a living cell. In this article, we summarize the most relevant tools that allow the swift assembly of DNA sequences into useful plasmids for biotechnological purposes. We cover the main components and stages in a typical DNA assembly workflow, namely in silico design, de novo gene synthesis, and in vitro and in vivo sequence assembly methodologies.

Escherichia coli with two linear chromosomes.

A number of attempts have been made to simplify the synthesis of whole chromosomes to generate artificial microorganisms. However, the sheer size of the average bacterial genome makes the task virtually impracticable. A major limitation is the maximum assembly DNA size imposed by the current available technologies. We propose to fragment the bacterial chromosome into autonomous replicating units so that (i) each episome becomes small enough to be assembled in its entirety within an assembly host and (ii) the complete episome set should be able to generate a viable cell. In this work, we used the telN/tos system of bacteriophage N1 to show that the circular genome of Escherichia coli can be split into two linear chromosomes that complement each other to produce viable cells.

Single step BP/LR combined Gateway reactions.

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.

Advanced DNA assembly technologies in drug discovery.

Recombinant DNA technologies have had a fundamental impact on drug discovery. The continuous emergence of unique gene assembly techniques resulted in the generation of a variety of therapeutic reagents such as vaccines, cancer treatment molecules and regenerative medicine precursors. With the advent of synthetic biology there is a growing need for precise and concerted assembly of multiple DNA fragments of various sizes, including chromosomes. In this article, we summarize the highlights of the recombinant DNA technology since its inception in the early 1970s, emphasizing on the most recent advances, and underscoring their principles, advantages and shortcomings. Current and prior cloning trends are discussed in the context of sequence requirements and scars left behind. Our opinion is that despite the remarkable progress that has enabled the generation and manipulation of very large DNA sequences, a better understanding of the cell's natural circuits is needed in order to fully exploit the current state-of-the-art gene assembly technologies.

Recombination-based DNA assembly and mutagenesis methods for metabolic engineering.

In recent years there has been a growing interest in the precise and concerted assembly of multiple DNA fragments of diverse sizes, including chromosomes, and the fine tuning of gene expression levels and protein activity. Commercial DNA assembly solutions have not been conceived to support the cloning of very large or very small genetic elements or a combination of both. Here we summarize a series of protocols that allow the seamless, simultaneous, flexible, and highly efficient assembly of DNA elements of a wide range of sizes (up to hundred thousand base pairs). The protocols harness the power of homologous recombination and are performed either in vitro or within the living cells. The DNA fragments may or may not share homology at their ends. An efficient site-directed mutagenesis protocol enhanced by homologous recombination is also described.

Genetic assembly tools for synthetic biology.

With the completion of myriad genome sequencing projects, genetic bioengineering has expanded into many applications including the integrated analysis of complex pathways, the construction of new biological parts and the redesign of existing, natural biological systems. All these areas require the precise and concerted assembly of multiple DNA fragments of various sizes, including chromosomes, and the fine-tuning of gene expression levels and protein activity. Current commercial cloning products are not robust enough to support the assembly of very large or very small genetic elements or a combination of both. In addition, current strategies are not flexible enough to allow further modifications to the original design without having to undergo complicated cloning strategies. Here, we present a set of protocols that allow the seamless, simultaneous, flexible, and highly efficient assembly of genetic material, designed for a wide size dynamic range (10s to 100,000s base pairs). The assembly can be performed either in vitro or within the living cells and the DNA fragments may or may not share homology at their ends. A novel site-directed mutagenesis approach enhanced by in vitro recombineering is also presented.

Characterizing diffusion dynamics of a membrane protein associated with nanolipoproteins using fluorescence correlation spectroscopy.

Nanolipoprotein particles (NLPs) represent a unique nanometer-sized scaffold for supporting membrane proteins (MP). Characterization of their dynamic shape and association with MP in solution remains a challenge. Here, we present a rapid method of analysis by fluorescence correlation spectroscopy (FCS) to characterize bacteriorhodopsin (bR), a membrane protein capable of forming a NLP complex. By selectively labeling individual components of NLPs during cell-free synthesis, FCS enabled us to measure specific NLP diffusion times and infer size information for different NLP species. The resulting bR-loaded NLPs were shown to be dynamically discoidal in solution with a mean diameter of 7.8 nm. The insertion rate of bR in the complex was ∼55% based on a fit model incorporating two separate diffusion properties to best approximate the FCS data. More importantly, based on these data, we infer that membrane protein associated NLPs are thermodynamically constrained as discs in solution, while empty NLPs appear to be less constrained and dynamically spherical.

Membrane protein expression: no cells required.

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).

Insertion of membrane proteins into discoidal membranes using a cell-free protein expression approach.

We report a cell-free approach for expressing and inserting integral membrane proteins into water-soluble particles composed of discoidal apolipoprotein-lipid bilayers. Proteins are inserted into the particles, circumventing the need of extracting and reconstituting the product into membrane vesicles. Moreover, the planar nature of the membrane support makes the protein freely accessible from both sides of the lipid bilayer. Complexes are successfully purified by means of the apoplipoprotein component or by the carrier protein. The method significantly enhances the solubility of a variety of membrane proteins with different functional roles and topologies. Analytical assays for a subset of model membrane proteins indicate that proteins are correctly folded and active. The approach provides a platform amenable to high-throughput structural and functional characterization of a variety of traditionally intractable drug targets.

Gateway(®) recombinational cloning: a biological operating system.

The Gateway(®) cloning system sets a new trend in molecular biology by addressing the difficulties of adaptability, efficiency and compatibility of the traditional cloning approaches. Based on the well-characterized site-specific recombination system of phage lambda, the Gateway(®) technology allows the cloning, combining and transferring of DNA segments between different expression platforms in a high-throughput manner while maintaining orientation and the reading frame of the fragment or fragments of interest. In this article, the key-aspects and potential applications of this system are reviewed. Benefits and weaknesses of this and other cloning technologies are discussed.