Mutation and screening to increase chymosin yield in a genetically-engineered strain ofAspergillus awamori

Summary Through the course of five rounds of mutagenesis of a genetically-engineered strain ofAspergillus awamori, the yield of a heterologous protein (the acid protease, calf chymosin) increased four-fold. This was accomplished through the use of an agar plate screen incorporating the colony restrictor 2,6-dichloro-4-nitroaniline (dichloran) and the acid protease inhibitor diazoacetyl-norleucine methyl ester (DAN) to reduce high background concentrations of the native acid protease. A miniaturized liquid culture growth method using 24-well culture plates was an intermediate screen between agar plate and shake flask cultures. Analysis of broth samples for active calf chymosin was accomplished with a highly specific, 96-well microtiter plate turbidimetric assay.     

Effects of Calcium on Sugar Transport in Azotobacter vinelandii

A fast and environmentally safe procedure was used to study sugar uptake by Azotobacter vinelandii. Transport experiments were performed in a 24-well plate and aerated by rapid oscillatory vibration. Samples were washed by centrifugation and dissolved in biodegradable scintillation cocktail for counting. At cell concentrations up to 6 × 10⁸ cells per ml, the uptake of sucrose was a function of time and was proportional to the cell concentration. This modified uptake assay was used to test the effect of cations on sugar uptake in A. vinelandii. Results showed that Ca²⁺ at 1 to 2 mM stimulated sucrose uptake by decreasing the apparent K_m of sucrose transport. Higher Ca²⁺ concentrations inhibited sucrose uptake in this organism.     

An automated filtration assay for protein kinase C ligands

[3H]Phorbol dibutyrate ([3H]PDBu) binding to soluble mouse brain protein kinase C (PKC) was established in a 96-well microtiter plate assay. [3H]PDBu-PKC receptor complexes were rapidly aspirated from wells, filtered, and washed onto glass fiber filter mats using an automated cell harvester. Results were compared to a modification of a previously described assay in which components were incubated in tubes, and manually delivered and washed onto filters with a manifold filtration apparatus. Both 96-well plate and tube assays gave qualitatively and quantitatively similar results since: (i) [3H]PDBu binding to PKC was phosphatidylserine (PS) dependent and calcium stimulatable; (ii) the amounts of [3H]PDBu bound by filters with each technique at receptors excess were similar, 3.2 +/- 0.3 and 3.1 +/- 0.4 pmol respectively; and (iii) the affinities of [3H]PDBu for PKC were comparable; Kd's were 1.95 +/- 0.3 and 2.2 +/- 0.55 nM, respectively. The 96-well plate assay was more accurate and rapid than the tube assay. The microtiter plate assay was adapted for use with [N,N-dimethyl-3H]N,N-dimethylstaurosporine ([3H]DMS). With [3H]PDBu and [3H]DMS as ligands, the 96-well plate method was used for the rapid discrimination of agents which bound selectively at the regulatory and/or catalytic domains of PKC.     

Investigation of high-throughput ultrafiltration for the determination of an unbound compound in human plasma using liquid chromatography and tandem mass spectrometry with electrospray ionization

A high-throughput ultrafiltration method with a direct injection assay has been developed to determine unbound concentrations of a high-protein binding compound, an alpha(v)beta(3) bone integrin antagonist (I), in human plasma for a clinical pharmacokinetic study. The 96-well MultiScreen filter plate with Ultracel-PPB membrane was evaluated for the separation of unbound from protein-bound compound I by ultrafiltration. The sample preparation was automated using a Packard MultiPROBE II EX liquid handling system to transfer the plasma samples to the 96-well PPB plate for centrifugation and to prepare ultrafiltrate samples for analysis. Using on-line extraction with a column-switching setup for sample clean-up and separation, the ultrafiltrate samples were directly injected onto a reversed-phase HPLC system and analyzed using a mass spectrometer interfaced with an electrospray ionization (ESI) source in the positive ionization mode (LC/ESI-MS/MS). The performance of the ultrafiltration using Ultracel-PPB 96-well plate for unbound I analysis was evaluated and optimized with respect to sample volume, centrifugation temperature, speed and time, and the relationship of the well positions of the PPB plate versus filtrate volumes and concentrations. The assay intraday accuracy and precision were between 93.9 and 104.8 and <7.3% (CV), respectively. The linear range of the calibration curve for the assay was 0.1-500 ng/mL on a Finnigan TSQ Quantum LC/ESI-MS/MS system. Evaluation and validation of the unbound plasma assay demonstrated it to be rapid, sensitive and reproducible.     

原代培养大鼠前列腺细胞建立前列腺增生筛药模型

目的建立原代培养大鼠前列腺上皮细胞体外筛药模型。方法无菌状态下取雄性SD大鼠腹侧叶前列腺,称量后,剪成1 mm3小块,经Ⅱ型胶原酶消化1 h后,过滤、离心获取前列腺细胞,接种于24孔板培养并对细胞进行形态学和免疫组织化学鉴定。获取的大鼠前列腺上皮细胞分别接种于96孔板和24孔板培养12 d后给予系列剂量(0.1～1000μmol/L)的他莫昔芬和阳性药物爱普列特处理72 h,利用CCK-8法检测前列腺上皮细胞存活率并计算药物IC50值,并进一步通过Giemsa染色后观察细胞生长及形态变化。结果细胞鉴定结果显示,获取的细胞具典型上皮细胞形态特征,细胞角蛋白和前列腺特异性抗原表达阳性,提示所获细胞为前列腺上皮细胞。爱普列特与前列腺上皮细胞孵育72 h后,CCK-8法检测结果显示能够明显抑制前列腺上皮细胞生长,其IC50值为42.7μM,进一步镜下观察结果显示,爱普列特未明显改变大鼠前列腺上皮细胞形态,但能导致存活细胞数减少。他莫昔芬则对大鼠前列腺上皮细胞生长无明显抑制作用,镜下观察前列腺上皮细胞数量和形态未见明显改变。结论利用原代培养SD大鼠前列腺上皮细胞可以成功建立前列腺增生体外筛选模型。     

Development of a semi-high-throughput growth assay for the filamentous actinobacteria <Emphasis Type="Italic">Frankia</Emphasis>

Filamentous bacteria pose unique challenges for testing multiple variables or growth parameters limiting the use of high-throughput methods. A semi-high-throughput growth assay system was developed to overcome these obstacles and validated for the filamentous actinobacteria Frankia. The 24-well plate assay was versatile for testing multiple growth medium parameters and provided reproducible results across wells and between plates. Under conditions of increased complexity, statistical analysis demonstrated that the variance was dependent on the experimental parameters and not the assay system. The 24-well plate assay was shown to be multipurpose for testing numerous variables on cell growth or other biological properties.     

Protein-free culture medium improvement: testing additives and their interactive effects in 96-well plates

In order to achieve a rational screening of additives suspected to improve antibody production over basal Dulbecco's Modified Eagle Medium (DMEM), we have developed a 96-well plate method for simultaneously testing individual and interactive effects of multiple additives. Cell viabilities were determined directly in each well by a colorimetric assay (MTT assay) and antibody production was measured by an enzyme-linked immunosorbent assay (ELISA) performed on well supernatants. Such as supplemented culture medium might considerably reduce production costs since commercial serum- or protein-free media are sold at serum-enriched basal medium costs. The CBM-P22 mouse cell line used in this study was shown to be sensitive to key amino acids, oxalacetic acid, ethanolamine and selenium at low cell density (<1 × 10⁵ cells·ml^–1). When these cells were inoculated in 96-well plates their antibody productivity was improved (sevenfold) by adding these additives to the basal DMEM as evidenced by ELISA absorbance readings. This improved productivity, obtained with the supplemented DMEM, named DOWSENs, is comparable to the one obtained with the commercial, but costly, protein-free hybridoma medium (PFHM II), taken here as the positive control. Results were confirmed by growing these cells in different media in standard (25 cm²) T-flask static culture. In addition, specific amino acid consumption, as analysed by HPLC, showed that asparagine and tryptophan when added to DMEM may improve antibody production, even if these amino acids are not limiting or highly consumed in PFHM II. Using this 96-well plate assay allows the assessment of a large number of different assays with a few milligrams of the product(s) tested. This is a rapid technique that gives results for additives that are not easily quantified by analytical techniques or for the study of interactive effects, which is time consuming and labour-intensive when done in individual T-flasks. Correspondence to: J. Côté     

Establishment of an efficient method to quantify embryo attachment to endometrial epithelial cell monolayers

During implantation, complex embryo-endometrium interactions result in blastocyst adhesion. To study the mechanisms of implantation, an effective assay for monitoring adhesiveness between embryos and endometrial epithelium is essential. In this study, we describe a simple and reliable method to quantify embryo-endometrium adhesion in vitro. Murine blastocysts or BeWo trophoblast spheroids were cocultured with monolayers of RL95-2 endometrial epithelial cells (EEC) grown in 96-well plates. At the end of coculture, the wells were filled with medium, and the plate was sealed with an adhesive film, inverted, and centrifuged at 25 x g for 5 min. After centrifugation, the plate was kept inverted and directly examined microscopically to determine whether the blastocysts or spheroids were attached to EEC monolayers. Our assay demonstrated that blastocysts recovered at 1200-1400 h on d 4 were more adherent to EEC than those recovered earlier, consistent with the timing of intrauterine embryo activation. Serum also enhanced blastocyst-EEC adhesion. Spheroid-EEC adhesion was inhibited by blocking Ca(2+) influx with extracellular Ca(2+) chelators (ethylenediaminetetraacetic acid or ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) or a Ca(2+) channel blocker (verapamil) but not by interfering with Ca(2+) release from intracellular stores using chelating (1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) or depleting (thapsigargin) agents. Using 96-well plates for coculture, centrifugation, and examination to minimize transfer procedures, our assay system is readily applicable to investigate implantation mechanisms.     

A simplified in vitro model of oxidant injury using vascular endothelial cells

Summary Oxidant injury of the vascular endothelium is considered an early event in the pathogenesis of atherosclerosis. The model of oxidant injury is crucial to the investigation of antioxidants. In the present study, a convenient in vitro model of oxidant injury induced by hydrogen peroxide (H₂O₂) was developed using bovine pulmonary artery endothelial cells (PAEC). Viability of PAEC grown in 96-well culture plates was determined with methylthiazol tetrazolium (MTT) colorimetric assay. Cell membrane integrity was measured by lactate dehydrogenase (LDH) release from PAEC grown in 24-well plates. Malondialdehyde (MDA, a product of lipid peroxidation) in PAEC grown in 6-well plates was detected by a thiobarbituric acid fluorometric assay. Incubation of H₂O₂ with PAEC caused a dose-dependent decrease of cell viability, an increase of LDH release, and an elevation of MDA production. MTT assay was convenient, quantitative, non-radioactive, and suitable for testing a large number of samples. The fluorometric assay for measuring MDA production in endothelial cells used 6-well plates instead of 80-cm² flasks employed by previous investigators. The use of multiwell culture plates in these assays made it possible for more samples to be tested in any single experiment. The three assays are reproducible with low intraplate and interplate coefficients of variation. This in vitro model is suitable for screening antioxidants and for studying pharmacodynamics at the cellular level.     

Anopheles stephensi and Toxorhynchites amboinensis: aseptic rearing of mosquito larvae on cultured cells

Aseptic larvae of Anopheles stephensi and Toxorhynchites amboinensis were reared on a continuous cell line (RU TAE 12 V) from the mosquito, T. amboinensis, that grew in suspension as multicellular vesicles. Surface-sterilized eggs were hatched in a 24-well plate containing 0.2 ml of Leibovitz's L-15 medium per well and incubated in a humidified atmosphere. Toxorhynchites amboinensis eggs of 36 hr or older were placed singly to assure hatching and avoid cannibalism. Hatching rates were over 80%. All larval instars were maintained in L-15 medium at 28 C with a 12-hr photoperiod. Anopheles stephensi larvae were reared in 25-cm2 tissue culture flasks containing 10 ml of L-15 medium with 30 to 50 first and second instar larvae or 10 third and fourth instar larvae per flask. Toxorhynchites amboinensis larvae remained in the 24-well plate in 1.5 ml of medium through the second instar; third instar larvae were kept in 12-well plates (3 ml of medium per well) and transferred to 25-cm2 flasks (10 ml per flask) when they reached the fourth instar. First and second instar A. stephensi larvae were fed cultured cells once, and third or fourth instar larvae twice a day. Toxorhynchites amboinensis larvae were fed vesicles once during the first 4 days after hatching, and every 1 or 2 days thereafter. Each A. stephensi larva consumed approximately 2 X 10(6) cells, and T. amboinensis larvae 10 times more cells before pupating. Anopheles stephensi pupated after 7 to 8 days and adults emerged during days 9 to 11. Pupation in T. amboinensis began on day 21 after hatching and adults emerged 5 days later. Cell lines isolated from A. stephensi larvae or embryos of the ticks Rhipicephalus sanguineus and Anocentor (Dermacentor) nitens supported only limited growth of A. stephensi larvae. Defibrinated hamster (Mesocricetus auratus) blood, though readily ingested, did not support the growth of A. stephensi whereas larvae reared on blood cells plus T. amboinensis cells showed limited growth.     

Simplification of aggregate culture of human mesenchymal stem cells as a chondrogenic screening assay

Aggregate culture provides a three-dimensional (3-D) environment for differentiating or differentiated cells; it is particularly useful to study in vitro chondrogenesis and cartilage biology. We have recently ported this method from a conical tube-based format to a 96-well plate format for the study of mesenchymal stem cell (MSC) chondrogenesis. The microplate format has greatly reduced the workload and materials cost, while maintaining reproducible chondrogenic differentiation. A long-term goal is to fully automate aggregate culture--this requires critically identifying all the indispensable steps of the protocol. Robotic laboratory equipment for manipulating microplate assays are commercially available; however centrifugation steps are difficult to implement automatically. We, therefore, tested whether the centrifugation step can be eliminated, thus significantly streamlining the assay workflow. By comparing aggregates prepared from human bone marrow-derived MSCs (hMSCs) that were formed either through centrifugation or through free sedimentation, we found that both methods produce aggregates with similar formation kinetics, and that there was no perceptible difference in the timing of the appearance of markers of chondrogenesis. Thus, it appears safe to eliminate the centrifugation step from the aggregate culture protocol. This results in significant time and effort savings and paves the way for future full automation of the aggregate assay.     

Expression of three reporter genes in four cell lines developed from <Emphasis Type="Italic">Papilio demoleus</Emphasis> Linnaeus (Lepidoptera: Papilionidae)

This paper used recombinant baculoviruses that carried three reporter genes, green fluorescent protein (GFP), β-galactosidase, and secreted alkaline phosphatase (SEAP), to infect four new cell lines from Papilio demoleus Linnaeus larvae (named RIRI-PaDe-1, RIRI-PaDe-2, RIRI-PaDe-3, and RIRI-PaDe-4). The expression levels of the three recombinant proteins were detected at 24, 48, 72, 96, 120, and 144 h after infection and compared with Sf9 and High Five cells to evaluate the characteristics of these four cell lines as host cells. The inoculation densities of the tested cell lines were 2?×?10⁴ cells/well (96-well plate) and 1?×?10⁵ cells/well (24-well plate), and adding a volume of virus stock resulted in an MOI of 5.0. The results showed that the four cell lines could be infected by recombinant baculovirus and that cell lysis occurred 96 h after infection. In the four tested cell lines, only a small number of RIRI-PaDe-1 and RIRI-PaDe-3 cells expressed recombinant GFP and showed green fluorescence. The expression was much lower than that of Sf9 and High Five. Comparing the intracellular and extracellular activity of β-galactosidase indicated that the P. demoleus cell system was more suitable for the expression of secreted proteins, and its extracellular β-galactosidase level was close to that of Sf9, but the expression level of SEAP was far lower than those of Sf9 and High Five.     

High-throughput purification of recombinant proteins using self-cleaving intein tags

High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (βGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format.     

Comparison between three rapid methods for direct diagnosis of influenza and the conventional isolation procedure.

Besides the rapid diagnostic tests based on influenza A and B antigens nucleoproteins detection, which are routinely used, the isolation of influenza strains is still required to obtain recent variant isolates for full antigenic characterization, in order to up-date the influenza vaccine composition. To increase the rapidity and the efficacy of the virus growth, we implemented a culture test in 24-well plates by centrifugation of samples on to LLCMK2 cells in the presence of trypsin. This test was routinely applied to 331 nasopharyngeal swabs collected during the influenza A outbreak in the winters 1988-1989 and to 962 in 1989-1990. The centrifugation culture assay has been compared with the direct detection of NP antigens in the clinical samples by immunofluorescence and capture ELISA tests and with the conventional virus isolation by inoculation of the samples to embryonated eggs and to LLCMK2 cell cultures. Compared with the NP antigen detection tests, the centrifugation culture assay closely correlated (r = 0.95) and the sensitivity and specificity were also excellent, 93.4% and 99.6%, respectively. Compared with the conventional culture assays, the centrifugation culture markedly increased the performance (five times) and rapidity (2 days) of influenza virus isolation and identification.     

A rapid and sensitive quantitative kinase activity assay using a convenient 96-well format

Activation of protein kinases in response to growth factor and extracellular matrix stimulation has been implicated in regulating a number of cell functions including differentiation, gene expression, migration, and proliferation. An improved quantitative assay for measuring protein kinase activity is crucial to the detailed study of this important category of signaling proteins and their role in regulating cell behavior. We describe a modified in vitro kinase activity assay that is both sensitive and quantitative. It offers several advantages when compared to the traditional immunoprecipitation/kinase assay: (i) high sensitivity that reduces the required amount of cell lysate by an order of magnitude, (ii) an immunoseparation technique utilizing antibody immobilization onto the surface of microtiter wells that replaces the cumbersome immunoprecipitation method, (iii) a 96-well plate configuration that eases handling of multiple samples and increases throughput of the assay, and (iv) the use of 96-well filter plates that greatly reduces radioactive liquid waste generation. While we implement this technique in a case study for measuring the activity of extracellular signal-regulated kinase 2 (ERK2), this assay can be extended to studying other protein kinases by using an appropriate antibody and in vitro substrate for the kinase of interest.     

Chemiluminometric biochemical induction assay (CBIA) for the detection of DNA-damaging agents

A microbroth chemiluminometric version of the biochemical induction assay (BIA) was developed using a chemiluminescent substrate widely used to detect beta-galactosidase in high-throughput screening (HTS) laboratories. The assay was run in both 96-well and 384-well plate formats using the Zymark RapidPlate liquid handling system to transfer samples and reagents. Chemiluminescence was read using the Victor-2 multilabel counter. The new microbroth chemiluminometric method, the CBIA, allowed rapid screening of samples, crude extracts, and pure compounds for their DNA-damaging effects in bacteria. In screening a small subset of our natural products library samples by the agar plate BIA and the CBIA, the latter yielded a higher hit rate, suggesting it is more sensitive than the agar plate assay. The CBIA was unaffected by the colored samples often encountered during screening of crude natural products extracts.     

Real-time immuno-polymerase chain reaction in a 384-well format: Detection of vascular endothelial growth factor and epidermal growth factor-like domain 7

Immuno-polymerase chain reaction (immuno-PCR) combines the specificity of antibodies with the amplification power of PCR to detect low levels of proteins. Here, we describe the development of a 384-well immuno-PCR method that uses streptavidin coated on a PCR plate to capture complexes of biotinylated capture antibody, antigen, and DNA-labeled detection antibody. Unbound molecules are removed by a wash step using a standard plate washer. Antibody–DNA molecules in bound complexes are then detected directly on the plate using real-time PCR. Circulating human vascular endothelial growth factor concentrations measured by this method correlated with measurements obtained from enzyme-linked immunosorbent assay (ELISA). Using this method, we developed an assay for human epidermal growth factor-like domain 7 (EGFL7), an extracellular matrix-bound angiogenic factor. EGFL7 is expressed at a higher level in certain cancers, although endogenous EGFL7 concentrations have not been reported. Our 384-well EGFL7 immuno-PCR assay can detect 0.51 pM EGFL7 in plasma, approximately 16-fold more sensitive than the ELISA, utilizing the same antibodies. This assay detected EGFL7 in lysates of non-small-cell lung cancer and hepatocellular carcinoma cell lines and also hepatocellular carcinoma, breast cancer, and ovarian cancer tissues. This 384-well immuno-PCR method can be used to develop high-throughput biomarker assays.     

Differential Centrifugation in Culture and Differentiation of Rat Neural Stem Cells

(1) The study of neural stem cells (NSC) has attracted much attention in recent years because of their therapeutic potential. However, the problem in culture and differentiation of NSC was how to obtain single cell suspension that preserves the function of NSC, and remove the debris caused by mechanical dissociation. In the present study, we try to find a simple and effective way to address the problem, i.e. differential centrifugation. (2) After a gentle mechanical dissociation using Pasteur pipette, the suspension was first centrifuged at 100 g for 5 min, and then recentrifuged at 400 g for 6 min. Finally, the two deposits were resuspended and seeded into culture flask respectively. The suspension from the second deposit was allowed for further culture and differentiation. Immunofluorescence technique was used to identify neural stem cell, neuron, astrocyte, and oligodendrocyte. (3) After the second differential centrifugation, single cell suspension was obtained with 2–3 cell clusters, and the cells not only grew to form neurospheres, but also differentiated into neurons, astrocytes, and oligodendrocytes. (4) Differential centrifugation is a simple and effective way to obtain single cell suspension, which will help make large-scale production of neurodifferentiated cells more effective.