A human cDNA library for high-throughput protein expression screening

We have constructed a human fetal brain cDNA library in an Escherichia coli expression vector for high-throughput screening of recombinant human proteins. Using robot technology, the library was arrayed in microtiter plates and gridded onto high-density filter membranes. Putative expression clones were detected on the filters using an antibody against the N-terminal sequence RGS-His(6) of fusion proteins. Positive clones were rearrayed into a new sublibrary, and 96 randomly chosen clones were analyzed. Expression products were analyzed by SDS-PAGE, affinity purification, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, and the determined protein masses were compared to masses predicted from DNA sequencing data. It was found that 66% of these clones contained inserts in a correct reading frame. Sixty-four percent of the correct reading frame clones comprised the complete coding sequence of a human protein. High-throughput microtiter plate methods were developed for protein expression, extraction, purification, and mass spectrometric analyses. An enzyme assay for glyceraldehyde-3-phosphate dehydrogenase activity in native extracts was adapted to the microtiter plate format. Our data indicate that high-throughput screening of an arrayed protein expression library is an economical way of generating large numbers of clones producing recombinant human proteins for structural and functional analyses.     

Improved protocols for the isolation and in-situ cryopreservation of cell colonies

Stable transfection and cloning of cells often require physical separation of cell colonies. In order to conveniently isolate cell clones from petri dishes, we developed a protocol starting with a soft agar overlay of cells. This reduces the risk of cell diffusion between different colonies. Cells from individual colonies are mechanically removed, incubated with trypsin, and cell suspensions are seeded onto parallel microtiter plates. The cell clones on one microtiter plate can be cryopreserved in situ using the protocol described here which was tested for a variety of cell lines. Replica plates can be used for screening and further expansion of interesting clones. If screening can also be performed in situ, e.g., by immunocytochemistry, immunofluorescence, or the polymerase chain reaction, it is possible to perform most steps necessary in cell cloning experiments on microtiter plates.     

A complete set of Escherichia coli open reading frames in mobile plasmids facilitating genetic studies.

To facilitate genetic studies of Escherichia coli, we constructed a complete set of mobile plasmid clones of intact open reading frames (ORFs). Their expression is strictly controlled by Ptac / lacI(q). The plasmids carrying each ORF were introduced into an F+ recA strain and stored in 96-well microtiter plates. In this way, 96 clones can be transferred simultaneously to F- bacteria using the conjugative system. This provides a convenient procedure for systematic identification of ORFs that suppress or complement mutations. We created two types of clone sets: the original set contained individual clones in 45 microtiter plates, and a second set contained pools of 48 clones stored in a single microtiter plate. Using these clone sets, we have identified 403 genes that can correct in trans the temperature-sensitive defect of cell division mutants, which would suggest multiple global regulators for bacterial cell division.     

Direct microtiter plate sequencing of PCR-amplified M13 clones from plaques using dried reagents.

A method is described using dried reagents in microtiter plates for direct PCR and sequencing of M13 plaques. The method has advantages of reliability and high throughput that make it suitable for medium to large-scale sequencing projects.     

Construction of a bovine yeast artificial chromosome (YAC) library

We have constructed a bovine yeast artificial chromosome (YAC) library to provide a common resource for bovine genome research. We used leukocytes of a Japanese black bull ( Bos taurus ) as the DNA source, AB1380 for the yeast host, and pYAC4 for the vector. The library consists of 24 576 clones arranged in 256 96-well microtiter plates. An average insert size estimated from the analysis of 251 randomly selected clones was 480 kb. The rate of chimeric YACs evaluated by fluorescence in situ hybridization (FISH) analysis of 44 randomly selected clones was 36·4%. To estimate the number of genome equivalents, PCR-based screening was performed with 48 primer pairs and isolated 3·2 clones on average. In order to provide broad access for the scientific community, this library has been incorporated into the Reference Library system which provides high density filters for colony hybridization screening and a common database of the library.     

Melon bacterial artificial chromosome (BAC) library construction using improved methods and identification of clones linked to the locus conferring resistance to melon Fusarium wilt (Fom-2).

Utilizing improved methods, two bacterial artificial chromosome (BAC) libraries were constructed for the multidisease-resistant line of melon MR-1. The HindIII library consists of 177 microtiter plates in a 384-well format, while the EcoRI library consists of 222 microtiter plates. Approximately 95.6% of the HindIII library clones contain nuclear DNA inserts with an average size of 118 kb, providing a coverage of 15.4 genome equivalents. Similarly, 96% of the EcoRI library clones contain nuclear DNA inserts with an average size of 114 kb, providing a coverage of 18.7 genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBac536 vector, and organellar DNA sequences. High-density filters were screened with two genetic markers FM and AM that co-segregate with Fom-2, a gene conferring resistance to races 0 and 1 of Fusarium wilt. Fourteen and 18 candidate BAC clones were identified for the FM and AM probes, respectively, from the HindIII library, while 34 were identified for the AM probe from filters A, B, and C of the EcoRI library.     

Equalizing growth in high‐throughput small scale cultivations via precultures operated in fed‐batch mode

An often underestimated problem when working with different clones in microtiter plates and shake flask screenings is the non‐parallel and non‐equal growth of batch cultures. These growth differences are caused by variances of individual clones regarding initial biomass concentration, lag‐phase or specific growth rate. Problems arising from unequal growth kinetics are different induction points in expression studies or uneven cultivation periods at the time of harvest. Screening for the best producing clones of a library under comparable conditions is thus often impractical or even impossible. A new approach to circumvent the problem of unequal growth kinetics of main cultures is the application of fed‐batch mode in precultures in microtiter plates and shake flasks. Fed‐batch operation in precultures is realized through a slow‐release system for glucose. After differently growing cultures turn to glucose‐limited growth, they all consume the same amount of glucose due to the fixed feed profile of glucose provided by the slow‐release system. This leads to equalized growth. Inherent advantages of this method are that it is easy to use and requires no additional equipment like pumps. This new technique for growth equalization in high‐throughput cultivations is simulated and verified experimentally. The growth of distinctly inoculated precultures in microtiter plates and shake flasks could be equalized for different microorganisms such as Escherichia coli and Hansenula polymorpha. Biotechnol. Bioeng. 2009;103: 1095–1102. © 2009 Wiley Periodicals, Inc.     

An Arrayed Bacteriophage P1 Genomic Library of Pneumocystis carinii

We have constructed an arrayed, large insert, multiple coverage genomic library of Pneumocystis carinii DNA using the bacteriophage P1 cloning system. The library consists of ∽4800 independent clones with an average insert size of ∽55 kbp individually arrayed in 50 microtiter plates, and is readily screened on ten or fewer microtiter plate-sized filters using a high density colony replicating device. Screening of the library for unique P. carinii sequences detected an average of 4–5 positive clones for each, consistent with a several-fold coverage of the ∽10-mbp P. carinii genome. Restriction and hybridization analyses demonstrated that the P1 clones in this library are quite stable and contain few, if any, chimeric inserts. Thus, this arrayed, large insert library off. carinii genomic DNA will be a valuable tool in the future genetic dissection of this important pathogen.     

From genes to proteins: high-throughput expression and purification of the human proteome

The development of high-throughput methods for gene discovery has paved the way for the design of new strategies for genome-scale protein analysis. Lawrence Livermore National Laboratory and Onyx Pharmaceuticals, Inc., have produced an automatable system for the expression and purification of large numbers of proteins encoded by cDNA clones from the IMAGE (Integrated Molecular Analysis of Genomes and Their Expression) collection. This high-throughput protein expression system has been developed for the analysis of the human proteome, the protein equivalent of the human genome, comprising the translated products of all expressed genes. Functional and structural analysis of novel genes identified by EST (Expressed Sequence Tag) sequencing and the Human Genome Project will be greatly advanced by the application of this high-throughput expression system for protein production. A prototype was designed to demonstrate the feasibility of our approach. Using a PCR-based strategy, 72 unique IMAGE cDNA clones have been used to create an array of recombinant baculoviruses in a 96-well microtiter plate format. Forty-two percent of these cDNAs successfully produced soluble, recombinant protein. All of the steps in this process, from PCR to protein production, were performed in 96-well microtiter plates, and are thus amenable to automation. Each recombinant protein was engineered to incorporate an epitope tag at the amino terminal end to allow for immunoaffinity purification. Proteins expressed from this system are currently being analyzed for functional and biochemical properties.     

Construction and Characterization of Two Bacterial Artificial Chromosome Libraries of Zhikong Scallop, Chlamys farreri Jones et Preston, and Identification of BAC Clones Containing the Genes Involved in Its Innate Immune System

Large-insert bacterial artificial chromosome (BAC) libraries are necessary for advanced genetics and genomics research. To facilitate gene cloning and characterization, genome analysis, and physical mapping of scallop, two BAC libraries were constructed from nuclear DNA of Zhikong scallop, Chlamys farreri Jones et Preston. The libraries were constructed in the BamHI and MboI sites of the vector pECBAC1, respectively. The BamHI library consists of 73,728 clones, and approximately 99% of the clones contain scallop nuclear DNA inserts with an average size of 110 kb, covering 8.0x haploid genome equivalents. Similarly, the MboI library consists of 7680 clones, with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1x. The combined libraries collectively contain a total of 81,408 BAC clones arrayed in 212 384-well microtiter plates, representing 9.1x haploid genome equivalents and having a probability of greater than 99% of discovering at least one positive clone with a single-copy sequence. High-density clone filters prepared from a subset of the two libraries were screened with nine pairs of Overgos designed from the cDNA or DNA sequences of six genes involved in the innate immune system of mollusks. Positive clones were identified for every gene, with an average of 5.3 BAC clones per gene probe. These results suggest that the two scallop BAC libraries provide useful tools for gene cloning, genome physical mapping, and large-scale sequencing in the species.     

From genes to proteins: High‐throughput expression and purification of the human proteome

The development of high‐throughput methods for gene discovery has paved the way for the design of new strategies for genome‐scale protein analysis. Lawrence Livermore National Laboratory and Onyx Pharmaceuticals, Inc., have produced an automatable system for the expression and purification of large numbers of proteins encoded by cDNA clones from the IMAGE (Integrated Molecular Analysis of Genomes and Their Expression) collection. This high‐throughput protein expression system has been developed for the analysis of the human proteome, the protein equivalent of the human genome, comprising the translated products of all expressed genes. Functional and structural analysis of novel genes identified by EST (Expressed Sequence Tag) sequencing and the Human Genome Project will be greatly advanced by the application of this high‐throughput expression system for protein production. A prototype was designed to demonstrate the feasibility of our approach. Using a PCR‐based strategy, 72 unique IMAGE cDNA clones have been used to create an array of recombinant baculoviruses in a 96‐well microtiter plate format. Forty‐two percent of these cDNAs successfully produced soluble, recombinant protein. All of the steps in this process, from PCR to protein production, were performed in 96‐well microtiter plates, and are thus amenable to automation. Each recombinant protein was engineered to incorporate an epitope tag at the amino terminal end to allow for immunoaffinity purification. Proteins expressed from this system are currently being analyzed for functional and biochemical properties. J. Cell. Biochem. 80:187–191, 2000. © 2000 Wiley‐Liss, Inc.     

Construction and characterization of a porcine P1-derived artificial chromosome (PAC) library covering 3.2 genome equivalents and cytogenetical assignment of six type I and type II loci

A porcine P1-derived artificial chromosome (PAC) library of a male German Landrace pig was constructed in pCYPAC2. In total 90,240 clones were generated and individually transferred into microtiter plates. An average insert size of 119.1 kb was determined by analyzing 150 randomly selected PAC clones by pulsed field electrophoresis, yielding approximately 3.2 genome equivalents. The stability of nine clones was followed through 110 generations showing no reduction of the insert size. The probability of identifying a specific chromosomal region within the library was tested by screening for the presence of seven type I and five type II loci. The analysis showed that most loci (10/12) were present in the library at least twice. To determine the percentage of chimerism, six clones were analyzed by fluorescence in situ hybridization (FISH) on metaphase chromosomes. We assign one type I locus (Triadin) and three type II loci (SW855, S0300, SW1129). Received: 24 November 1998 / Accepted: 1 February 1999     

Construction and Characterization of a Bacterial Artificial Chromosome (BAC) Library of Pacific White Shrimp, Litopenaeus vannamei

The pacific white shrimp (Litopenaeus vannamei) is one of the most economically important marine aquaculture species in the world. To facilitate gene cloning and characterization, genome analysis, physical mapping, and molecular selection breeding of marine shrimp, we have developed the techniques to isolate high-quality megabase-sized DNA from hemocyte nuclear DNA of female shrimp and constructed a bacterial artificial chromosome (BAC) genomic library for the species. The library was constructed in the Hind III site of the vector pECBAC1, consisting of 101,760 clones arrayed in 265 384-well microtiter plates, with an average insert size of 101 kb, and covering the genome approximately fivefold. To characterize the library, 92,160 clones were spotted onto high-density nylon filters for hybridization screening. A set of 18 pairs of overgo probes designed from eight cDNA sequences of L. vannamei genes were used in hybridization screening, and 35 positive clones were identified. These results suggest that the shrimp BAC libraries will provide a useful resource for screening of genomic regions of interest candidate genes, gene families, or large-sized synthetic DNA region and promote future works on comparative genomics, physical mapping, and large-scale genome sequencing in the species.     

High-throughput screening for gene libraries expressing carbohydrate hydrolase activity

A simple and fast method is described allowing screening of large number of Escherichia coli clones (4000 per day) for the presence of functional or improved carbohydrate hydrolase enzymes. The procedure is relatively cheap and has the advantage that carbohydrate degrading activity can be directly measured using liquid cultures grown in microtiter plates without the need of separation or purification steps.     

Magnetic bead purification of M13 DNA sequencing templates.

A method for the preparation of multiple M13 DNA sequencing templates is described. No phenol extraction is required and the procedure can be carried out in Eppendorf tubes or 96-well microtiter plates. Starting with a phage supernatant, the entire procedure is carried out in the same reaction vessel and all separation steps are based on a novel application of magnetic bead separation. The design of a 96-well magnetic separator is presented and the application of the method for large-scale sequencing is discussed.     

Construction and preliminary analysis of the ICRF human P1 library

P1 clone libraries have now been established as effective complements to cosmid and yeast artificial chromosome libraries in long-range mapping projects. To allow general access to P1 clones, we have constructed human and mouse P1 libraries. Clones have been picked into microtiter plates and used to prepare high-density filter grids, providing an efficient and easy screening system. Filters are being made available to other laboratories through the Reference Library System. In this work, we have developed a reliable protocol for generating P1 clones, based on the use of pulsed-field gel electrophoresis for size selection of DNA. A 1.2× genome coverage human library has been produced using this method. A preliminary analysis of this library is described.     

Rapid detection of salmonellae by immunoassays with titanous hydroxide as the solid phase

Radioimmunometric and enzyme-immunometric assays were developed for the detection of salmonellae in pure and mixed cultures as well as in 59 food samples. The performances of titanous hydroxide suspension and microtiter plates as the solid phase for the immobilization of microorganisms were compared in these immunoassays. Detection of populations of salmonella cells in pure culture, diluted with saline, was 4- to 10-fold more sensitive with the microtiter plates. However, with mixed culture of salmonella and other enterobacterial species, the detection sensitivity with titanous hydroxide was 100- to 160-fold more sensitive than with microtiter plates. Good correlation existed between results of a standard cultural method for the detection of salmonellae in foods and those obtained from radioimmunometric and enzyme-immunometric assays utilizing titanous hydroxide. However, a high incidence of false-positive and false-negative results with food samples occurred with the enzyme-immunometric assay utilizing microtiter plates. The results provided strong evidence for the merits of substituting titanous hydroxide for microtiter plates as the solid phase for the immobilization of salmonellae for their detection by immunoassays. The immunoassays were rapid and enabled the analysis of a large number of selective enrichment cultures of food samples for salmonellae within 8 h.     

Rapid detection of salmonellae by immunoassays with titanous hydroxide as the solid phase.

Radioimmunometric and enzyme-immunometric assays were developed for the detection of salmonellae in pure and mixed cultures as well as in 59 food samples. The performances of titanous hydroxide suspension and microtiter plates as the solid phase for the immobilization of microorganisms were compared in these immunoassays. Detection of populations of salmonella cells in pure culture, diluted with saline, was 4- to 10-fold more sensitive with the microtiter plates. However, with mixed culture of salmonella and other enterobacterial species, the detection sensitivity with titanous hydroxide was 100- to 160-fold more sensitive than with microtiter plates. Good correlation existed between results of a standard cultural method for the detection of salmonellae in foods and those obtained from radioimmunometric and enzyme-immunometric assays utilizing titanous hydroxide. However, a high incidence of false-positive and false-negative results with food samples occurred with the enzyme-immunometric assay utilizing microtiter plates. The results provided strong evidence for the merits of substituting titanous hydroxide for microtiter plates as the solid phase for the immobilization of salmonellae for their detection by immunoassays. The immunoassays were rapid and enabled the analysis of a large number of selective enrichment cultures of food samples for salmonellae within 8 h.     

Characterization of clones of HIV-1 infected HuT 78 cells defective in gag gene processing and of SIV clones producing large amounts of envelope glycoprotein

Single-cell clones of HIV-1 (FRE-3) or SIV/Mne infected HuT 78 cells were obtained by plating dilutions of virally infected HuT 78 cells on a monolayer of sheep choroid plexus cells in 96-well microtiter plates. Several of these clones produce HIV-1 virus mutants that accumulate the gag precursor polyprotein and lack a functional protease. These protease-deficient viruses are non-infectious and consist of aberrant "immature" virus particles as determined by electron microscopy. Several SIV mutants are also described that produce large amounts of either the envelope glycoprotein gp120 or the nucleic acid binding gag protein. These mutants are useful for the purification of these retroviral proteins, in developing assays of protease inhibitors, and in preparing SIV envelope protein vaccines.     

Automated Sanger dideoxy sequencing reaction protocol

The protocol for Sanger dideoxy chain termination reactions in DNA sequencing is tedious and prone to errors due to the repetitive character of the pipetting steps. An industrial robot, with the addition of a few simple parts, was programmed to automate the dideoxy sequencing reactions. The system is set up in a short time for routine operation and it is faster and more reliable than a human operator. It is flexible and allows variations and optimization of the standard procedure. Disposable microtiter plates at a controlled temperature are used. In one reaction cycle (about 50 min) up to 48 templates are processed. Up to 450 bases were resolved in automated DNA sequencing on samples prepared by the robot. The protocol is applicable to fluorescent as well as to radioactive labeling.