Large-scale T-DNA mutagenesis in Arabidopsis for functional genomic analysis

In planta Agrobacterium-mediated transformation combined with a soil-based herbicide selection for transgenic plants was used to recover large numbers of transgenic Arabidopsis plants for functional genomic studies. A tissue-culture-free system for generating transgenic plants was achieved by infiltrating Arabidopsis plants with Agrobacterium tumefaciens harboring a binary T-DNA vector containing the phosphinothricin acetyltransferase gene from Streptomyces hygroscopicus, and by selecting transgenic Arabidopsis growing in soil by foliar application of the herbicide Finale (phosphinothricin). Analysis of herbicide-resistant plants indicated that all were transgenic and that the T-DNA transformation process occurred late during flower development, resulting in a preponderance of independently derived T-DNA insertions. T-DNA insertions were usually integrated in a concatenated, rearranged form, and using linkage analysis, we estimated that T1 plants carried between one and five T-DNA loci. Using pooling strategies, both DNA and seed pools were generated from about 38,000 Arabidopsis plants representing over 115,000 independent T-DNA insertions. We show the utility of these transgenic lines for identifying insertion mutations using gene sequence and PCR-based screening. Electronic Publication     

'PACLIMS': A component LIM system for high-throughput functional genomic analysis

Background

Recent advances in sequencing techniques leading to cost reduction have resulted in the generation of a growing number of sequenced eukaryotic genomes. Computational tools greatly assist in defining open reading frames and assigning tentative annotations. However, gene functions cannot be asserted without biological support through, among other things, mutational analysis. In taking a genome-wide approach to functionally annotate an entire organism, in this application the ~11,000 predicted genes in the rice blast fungus (Magnaporthe grisea), an effective platform for tracking and storing both the biological materials created and the data produced across several participating institutions was required.

Results

The platform designed, named PACLIMS, was built to support our high throughput pipeline for generating 50,000 random insertion mutants of Magnaporthe grisea. To be a useful tool for materials and data tracking and storage, PACLIMS was designed to be simple to use, modifiable to accommodate refinement of research protocols, and cost-efficient. Data entry into PACLIMS was simplified through the use of barcodes and scanners, thus reducing the potential human error, time constraints, and labor. This platform was designed in concert with our experimental protocol so that it leads the researchers through each step of the process from mutant generation through phenotypic assays, thus ensuring that every mutant produced is handled in an identical manner and all necessary data is captured.

Conclusion

Many sequenced eukaryotes have reached the point where computational analyses are no longer sufficient and require biological support for their predicted genes. Consequently, there is an increasing need for platforms that support high throughput genome-wide mutational analyses. While PACLIMS was designed specifically for this project, the source and ideas present in its implementation can be used as a model for other high throughput mutational endeavors.     

Bacterial cell-free system for high-throughput protein expression and a comparative analysis of Escherichia coli cell-free and whole cell expression systems

Sixty-three proteins of Pseudomonas aeruginosa in the size range of 18-159 kDa were tested for expression in a bacterial cell-free system. Fifty-one of the 63 proteins could be expressed and partially purified under denaturing conditions. Most of the expressed proteins showed yields greater than 500 ng after a single affinity purification step from 50 microl in vitro protein synthesis reactions. The in vitro protein expression plus purification in a 96-well format and analysis of the proteins by SDS-PAGE were performed by one person in 4 h. A comparison of in vitro and in vivo expression suggests that despite lower yields and less pure protein preparations, bacterial in vitro protein expression coupled with single-step affinity purification offers a rapid, efficient alternative for the high-throughput screening of clones for protein expression and solubility.     

A less laborious approach to the high-throughput production of recombinant proteins in Escherichia coli using 2-liter plastic bottles

Contemporary approaches to biology often call for the high-throughput production of large amounts of numerous proteins for structural or functional studies. Even with the highly efficient protein expression systems developed in Escherichia coli, production of these proteins is laborious and time-consuming. We have simplified established protocols by the use of disposable culture vessels: common 2-liter polyethylene terephthalate beverage bottles. The bottles are inexpensive, fit conveniently in commonly available flask holders, and, because they are notched, provide sufficient aeration to support the growth of high-density cultures. The use of antibiotics and freshly prepared media alleviates the need for sterilization of media and significantly reduces the labor involved. Uninoculated controls exhibited no growth during the time required for protein expression in experimental cultures. The yield, solubility, activity, and pattern of crystallization of proteins expressed in bottles were comparable to those obtained under conventional culture conditions. After use, the bottles are discarded, reducing the risk of cross-contamination of subsequent cultures. The approach appears to be suitable for high-throughput production of proteins for structural or functional studies.     

High-throughput analysis of gene expression on tissue sections by in situ hybridization

Genome-scale sequencing projects, high-throughput RNAi screens, systematic gene targeting, and system-biology-based network predictions all depend on a validation of biological significance in order to understand the relevance of a particular finding. Such validation, for the most part, rests on low-throughput technologies. This article provides protocols that, in combination with suitable instrumentation, make possible a semi-automated analysis of gene expression on tissue sections by means of in situ hybridization. Knowledge of gene expression localization has the potential to aid, and thereby accelerate, the validation of gene functions.     

Construction of an efficient Escherichia coli cell‐free system for in vitro expression of several kinds of proteins

Cell‐free protein synthesis systems have offered several advantages over traditional cell‐based expression methods. In this study, the effects of extract preparation and an energy‐regenerating system on protein synthesis were investigated in an Escherichia coli cell‐free system. The results indicated that the expression level of enhanced green fluorescent protein (eGFP) with the S12 extract was higher than that with the S30 extract. Among four adenosine triphosphate‐regenerating sources, the cAMP/CP/CK system (including cAMP, creatine phosphate, and creatine kinase) proved to be the most efficient one to support high‐level expression of eGFP. Further studies showed that this established cell‐free system could be successfully used to produce one model protein (eGFP), two human proteins (AK2 and coenzyme synthase) and two membrane proteins (subunit b of F₁F₀ adenosine triphosphate synthase and aquaporin Z). This outcome will be helpful to develop the highly efficient cell‐free technology for the production of various proteins with different bio‐origins.     

A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell‐free protein synthesis systems

Cell‐free protein synthesis (CFPS) systems allow for robust protein expression with easy manipulation of conditions to improve protein yield and folding. Recent technological developments have significantly increased the productivity and reduced the operating costs of CFPS systems, such that they can compete with conventional in vivo protein production platforms, while also offering new routes for the discovery and production of biotherapeutics. As cell‐free systems have evolved, productivity increases have commonly been obtained by addition of components to previously designed reaction mixtures without careful re‐examination of the essentiality of reagents from previous generations. Here we present a systematic sensitivity analysis of the components in a conventional Escherichia coli CFPS reaction mixture to evaluate their optimal concentrations for production of the immunoglobulin G trastuzumab. We identify eight changes to the system, which result in optimal expression of trastuzumab. We find that doubling the potassium glutamate concentration, while entirely eliminating pyruvate, coenzyme A, NAD, total tRNA, folinic acid, putrescine and ammonium glutamate, results in a highly productive cell‐free system with a 95% reduction in reagent costs (excluding cell‐extract, plasmid, and T7 RNA polymerase made in‐house). A larger panel of other proteins was also tested and all show equivalent or improved yields with our simplified system. Furthermore, we demonstrate that all of the reagents for CFPS can be combined in a single freeze‐thaw stable master mix to improve reliability and ease of use. These improvements are important for the application of the CFPS system in fields such as protein engineering, high‐throughput screening, and biotherapeutics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:823–831, 2015     

Enzyme Free Cloning for high throughput gene cloning and expression

Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning (EFC) procedure, a PCR-only method that eliminates all variables other than PCR efficiency by circumventing enzymatic treatments. We compared the cloning efficiency of EFC with that of Ligation Independent Cloning (LIC). Both methods are well suited for HTP cloning, but EFC yields three times more transformants and a cloning efficiency of 91%, comparable with recombinational cloning methods and significantly better than LIC (79%). EFC requires only nanogram amounts of both vector and insert, does not require highly competent cells and is, in contrast to LIC, largely insensitive to variations in PCR product concentration. Automated protein expression screening of expression strains directly transformed with EFC reactions showed, that the traditional preceding step via a cloning strain can be circumvented. EFC proves an efficient and robust HTP cloning method, that is compatible with existing Ligation Independent Cloning vectors, and highly suitable for automation.     

Protein surface analysis for function annotation in high-throughput structural genomics pipeline

Structural genomics (SG) initiatives are expanding the universe of protein fold space by rapidly determining structures of proteins that were intentionally selected on the basis of low sequence similarity to proteins of known structure. Often these proteins have no associated biochemical or cellular functions. The SG success has resulted in an accelerated deposition of novel structures. In some cases the structural bioinformatics analysis applied to these novel structures has provided specific functional assignment. However, this approach has also uncovered limitations in the functional analysis of uncharacterized proteins using traditional sequence and backbone structure methodologies. A novel method, named pvSOAR (pocket and void Surface of Amino Acid Residues), of comparing the protein surfaces of geometrically defined pockets and voids was developed. pvSOAR was able to detect previously unrecognized and novel functional relationships between surface features of proteins. In this study, pvSOAR is applied to several structural genomics proteins. We examined the surfaces of YecM, BioH, and RpiB from Escherichia coli as well as the CBS domains from inosine-5'-monosphate dehydrogenase from Streptococcus pyogenes, conserved hypothetical protein Ta549 from Thermoplasm acidophilum, and CBS domain protein mt1622 from Methanobacterium thermoautotrophicum with the goal to infer information about their biochemical function.     

Rapid expression of vaccine proteins for B-cell lymphoma in a cell-free system

The idiotype (Id)-granulocyte-macrophage colony-stimulating factor (GM-CSF) fusion proteins are potential vaccines for immunotherapy of B-cell lymphoma. In this study, four vaccine candidates were constructed by fusing murine GM-CSF to the amino- or carboxy-terminus of the 38C13 murine B-lymphocyte Id scFv with two different arrangements of the variable regions of the heavy chain and light chain (VL-VH and VH-VL). scFv (VH-VL) and GM-CSF/scFv fusion proteins were expressed in an Escherichia coli cell-free protein synthesis system. In order to promote disulfide bond formation during cell-free expression, cell extract was pretreated with iodoacetamide (IAM), and a sulfhydryl redox buffer composed of oxidized and reduced glutathione was added. The E. coli periplasmic disulfide isomerase, DsbC, was also added to rearrange incorrectly formed disulfide linkages. The 38C13 B-lymphocyte Id scFv was expressed with 30% of its soluble yield in active form (43 microg/ml) when tested with an anti-idiotypic mAb, S1C5, as the capture antibody in radioimmunoassay. It was found that the amino-terminal GM-CSF fusion proteins, GM-VL-VH and GM-VH-VL, showed much higher activity than the carboxy-terminal GM-CSF fusion proteins, VL-VH-GM and VH-VL-GM, in stimulating the cell proliferation of a GM-CSF-dependent cell line, NFS-60. Between the two amino-terminal GM-CSF fusion proteins, GM-VL-VH showed a higher total and soluble yield than GM-VH-VL.     

Synthesis of membrane proteins in eukaryotic cell‐free systems

Cell‐free protein synthesis (CFPS) is a valuable method for the fast expression of difficult‐to‐express proteins as well as posttranslationally modified proteins. Since cell‐free systems circumvent possible cytotoxic effects caused by protein overexpression in living cells, they significantly enlarge the scale and variety of proteins that can be characterized. We demonstrate the high potential of eukaryotic CFPS to express various types of membrane proteins covering a broad range of structurally and functionally diverse proteins. Our eukaryotic cell‐free translation systems are capable to provide high molecular weight membrane proteins, fluorescent‐labeled membrane proteins, as well as posttranslationally modified proteins for further downstream analysis.     

Tandem Cell‐Free Protein Synthesis as a Tool for Rapid Screening of Optimal Molecular Chaperones

A simple and flexible method is developed for rapid screening of molecular chaperones that enhance the functional expression of recombinant proteins. A panel of molecular chaperones are transiently expressed in a reaction mixture of cell‐free protein synthesis and then a target protein is subsequently expressed in the presence of these presynthesized molecular chaperones. The biological activity of the cell‐free synthesized target protein is compared to identify the effective molecular chaperones. This strategy successfully identifies individual and combinations of bacterial molecular chaperones that markedly improved the functional expression of horseradish peroxidase. The authors believe that the presented strategy provides a versatile platform for the optimal production of functional proteins, and can also be extended to studies of other interacting proteins.     

A naturally enhanced green fluorescent protein from magnificent sea anemone (Heteractis magnifica) and its functional analysis

A novel fluorescent protein termed hmGFP homologous to the green fluorescent protein (GFP) from Aequorea victoria was cloned from the tentacles of sea anemone Heteractis magnifica by EST sequencing and analysis of cDNA library and followed by using RT-PCR. The sequence analysis suggested that the chromophore, consensus amino acids, and secondary structure of 11 beta-strands of hmGFP were similar to those of GFP from other species. The recombinant hmGFP protein with high purity was obtained by the fusion expression of pETTRX-hmGFP in Escherichia coli and subsequent purification. The pH sensitivity and fluorescence spectroscopy of recombinant hmGFP were characterized. The excitation spectrum of recombinant hmGFP has a rather wide major peak with a maximum at 490 nm and a shoulder at 420 nm, and its emission spectrum at 510 nm. The expression of hmGFP and the chimera IPL through hmGFP in CHO cells has shown that the fusion protein IPL through hmGFP has retained the normal membrane targeting of the IPL from Dasyatis akajei, as well as maintaining fluorescent properties similar to those of native hmGFP, suggesting a promising prospect of the application in biotechnology research for the new protein.     

An expression vector tailored for large-scale, high-throughput purification of recombinant proteins

Production of milligram quantities of numerous proteins for structural and functional studies requires an efficient purification pipeline. We found that the dual tag, his(6)-tag-maltose-binding protein (MBP), intended to facilitate purification and enhance proteins' solubility, disrupted such a pipeline, requiring additional screening and purification steps. Not all proteins rendered soluble by fusion to MBP remained soluble after its proteolytic removal, and in those cases where the protein remained soluble, standard purification protocols failed to remove completely the stoichiometric amount of his(6)-tagged MBP generated by proteolysis. Both liabilities were alleviated by construction of a vector that produces fusion proteins in which MBP, the his(6)-tag and the target protein are separated by highly specific protease cleavage sites in the configuration MBP-site-his(6)-site-protein. In vivo cleavage at the first site by co-expressed protease generated untagged MBP and his(6)-tagged target protein. Proteins not truly rendered soluble by transient association with MBP precipitated, and untagged MBP was easily separated from the his-tagged target protein by conventional protocols. The second protease cleavage site allowed removal of the his(6)-tag.     

RETRACTED ARTICLE: Virus-induced gene silencing for functional analysis of selected genes

Virus-induced gene silencing (VIGS) is a technology that exploits an RNA-mediated antiviral defense mechanism and has been shown to be of great potential in plant reverse genetics. Circumvention of plant transformation, methodological simplicity, robustness, and speedy results makes VIGS an attractive alternative instrument in functional genomics, even in a high throughput fashion. The system is well established in Nicotiana benthamiana, and efforts are being made to improve VIGS in other species, including monocots. Here, we discuss the issues specific to the application of VIGS technology to determine gene function, which has revealed the roles of a variety of genes in disease resistance, abiotic stress, cellular signaling and secondary metabolite biosynthesis. M. R. Godge and A. Purkayastha made equal contributions and hence should be treated as joint first authors for this paper.     

Cell‐free translational screening of an expression sequence tag library of Clonorchis sinensis for novel antigen discovery

The rapidly evolving cloning and sequencing technologies have enabled understanding of genomic structure of parasite genomes, opening up new ways of combatting parasite‐related diseases. To make the most of the exponentially accumulating genomic data, however, it is crucial to analyze the proteins encoded by these genomic sequences. In this study, we adopted an engineered cell‐free protein synthesis system for large‐scale expression screening of an expression sequence tag (EST) library of Clonorchis sinensis to identify potential antigens that can be used for diagnosis and treatment of clonorchiasis. To allow high‐throughput expression and identification of individual genes comprising the library, a cell‐free synthesis reaction was designed such that both the template DNA and the expressed proteins were co‐immobilized on the same microbeads, leading to microbead‐based linkage of the genotype and phenotype. This reaction configuration allowed streamlined expression, recovery, and analysis of proteins. This approach enabled us to identify 21 antigenic proteins. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:832–837, 2017     

A two-host fosmid system for functional screening of (meta)genomic libraries from extreme thermophiles

A new cloning system is described, which allows the construction of large-insert fosmid libraries in Escherichia coli and the transfer of the recombinant libraries to the extreme thermophile Thermus thermophilus via natural transformation. Libraries are established in the thermophilic host by site-specific chromosomal insertion of the recombinant fosmids via single crossover or double crossover recombination at the T. thermophilus pyr locus. Comparative screening of a fosmid library constructed from genomic DNA from the thermophilic spirochaete, Spirochaeta thermophila, for clones expressing thermoactive xylanase activity revealed that 50% of the fosmids that conferred xylanase activity upon the corresponding T. thermophilus transformants did not give rise to xylanase-positive E. coli clones, indicating that significantly more S. thermophila genes are functionally expressed in T. thermophilus than in E. coli. The novel T. thermophilus host/vector system may be of value for the construction and functional screening of recombinant DNA libraries from individual thermophilic or extremely thermophilic organisms as well as from complex metagenomes isolated from thermophilic microbial communities.