o-nitrophenyl-β-D-galactopyranoside Research Articles

Characterization of an Unusual Cold-Active β-Glucosidase Belonging to Family 3 of the Glycoside Hydrolases from the Psychrophilic Isolate Paenibacillus sp. Strain C7

We selected for spore-forming psychrophilic bacteria able to use lactose as a carbon source and one isolate, designated Paenibacillus sp. strain C7, that was phylogenetically related to, but distinct from both Paenibacillus macquariensis and Paenibacillus antarcticus. Some Escherichia coli transformants obtained with genomic DNA from this isolate hydrolyzed X-Gal (5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside) only below 30 degrees C, an indication of cold-active beta-galactosidase activity. Sequencing of the cloned insert revealed an open reading frame encoding a 756-amino acid protein that, rather than belonging to a family typically known for beta-galactosidase activity, belonged to glycoside hydrolase family 3, a family of beta-glucosidases. Because of this unusual placement, the recombinant enzyme (BglY) was purified and characterized. Consistent with its classification, the enzyme had seven times greater activity with the glucoside substrate ONPGlu (o-nitrophenyl-beta-D-glucopyranoside) than with the galactoside substrate ONPGal (o-nitrophenyl-beta-D-galactopyranoside). In addition, the enzyme had, with ONPGlu, a thermal optimum around 30 to 35 degrees C, activity over a broad pH range (5.5 to 10.9), and an especially low Km (<0.003 mM). Further examination of substrate preference showed that the BglY enzyme also hydrolyzed other aryl-beta-glucosides such as helicin, MUG (4-methylumbelliferyl-beta-D-glucopyranoside), esculin, indoxyl-beta-D-glucoside (a natural indigo precursor), and salicin, but had no activity with glucosidic disaccharides or lactose. These characteristics and substrate preferences make the BglY enzyme unique among the family 3 beta-glucosidases. The hydrolysis of a variety of aryl-beta-glucosides suggests that the enzyme may allow the organism to use these substrates in the environment and that its low Km on indoxyl-beta-D-glucoside may make it useful for producing indigo.

Prevalence of Staphylococcus aureus carriage by young Malaysian footballers during indoor training

Background: Research has shown that athletes are carrriers of Staphylococcus aureus during physical activity. Objective: To estimate the mean total plate count of S aureus carrried by footballers before and...

Enzyme immobilization in novel alginate–chitosan core-shell microcapsules

Alginate–chitosan core-shell microcapsules were prepared in order to develop a biocompatible matrix for enzyme immobilization, where the protein is retained either in a liquid or solid core and the shell allows permeability control over substrates and products. The permeability coefficients of different molecular weight compounds (vitamin B2, vitamin B12, and myoglobin) were determined through sodium tripolyphosphate (Na-TPP)-crosslinked chitosan membrane. The microcapsule core was formed by crosslinking sodium alginate with either calcium or barium ions. The crosslinked alginate core was uniformly coated with a chitosan layer and crosslinked with Na-TPP. In the case of calcium alginate, the phosphate ions of Na-TPP were able to extract the calcium ions from alginate and liquefy the core. A model enzyme, β-galactosidase, was immobilized in the alginate core and the catalytic activity was measured with o-nitrophenyl- β- d-galactopyranoside (ONPG). Change in the activity of free and immobilized enzyme was determined at three different temperatures. Na-TPP crosslinked chitosan membranes were found to be permeable to solutes of up to 17,000 Da molecular weight. The enzyme loading efficiency was higher in the barium alginate core (100%) as compared to the calcium alginate core (60%). The rate of ONPG conversion to o-nitrophenol was faster in the case of calcium alginate–chitosan microcapsules as compared to barium alginate–chitosan microcapsules. Barium alginate–chitosan microcapsules, however, did improve the stability of the enzyme at 37°C relative to calcium alginate–chitosan microcapsules or free enzyme. This study illustrates a new method of enzyme immobilization for biotechnology applications using liquid or solid core and shell microcapsule technology.

Enzymatic synthesis of galactosyl–xylose by Aspergillus oryzae β-galactosidase

In aqueous medium, the reaction catalyzed by Aspergillus oryzae β -galactosidase with O-nitrophenyl-β-d-galactopyranoside (ONPG) in the presence of an acceptor leads to the synthesis of transglycosylation compounds in addition to the hydrolysis products (ONP and galactose). Our goal was to develop a simple system for the synthesis of galactosyl–xylose, a disaccharide of possible application to diagnostics. To maximize synthesis yields, we have studied the effect of several conditions: increase of acceptor concentration (0.05–2.7M xylose), organic co-solvents (dimethylformamide, acetone) and reaction time.In the absence of co-solvents ONPG was completely consumed in 2h; with 0.5 M xylose the maximum yield of galactosyl–xylose (16%) was attained at 60min, while with 2.7M xylose the yield reached 21%. Both co-solvents tested decreased the kinetics of ONPG convertion into products and 50% (v/v) dimethylformamide was deleterious to the synthesis. However, in 50% (v/v) acetone the synthesis yield was 12% and interestingly, the proportion of transglycosylation with respect to the reacted substrate was higher than in buffer.The synthesis of galactosyl–ethyleneglycol was also studied; it was achieved with extremely high yield and no detectable hydrolysis products. This proves that other acceptor alcohols can be preferred over water in some conditions.

Peptide-Retargeted Adenovirus Encoding a Tissue Inhibitor of Metalloproteinase-1 Decreases Restenosis after Intravascular Gene Transfer

In this study we have attached cyclic targeting peptides by way of a poly-lysine spacer on the surface of an adenovirus using a transglutaminase enzymatic reaction to enhance transduction efficiency and to modify tissue tropism in vivo. Nuclear targeted lacZ- and TIMP-1-encoding adenoviruses were coupled to a peptide-motif (HWGF) that can bind to matrix metalloproteinase (MMP)-2 and MMP-9. Modified viruses were used to evaluate gene transfer efficiency, biodistribution, and the effect on neointima formation following balloon denudation injury. In vitro, both rabbit aortic smooth muscle cells and human endothelial hybridoma cells demonstrated significantly increased reporter gene expression with HWGF-modified adenoviruses (AdlacZ(HWGF)) compared with control (AdlacZ) or mismatch peptide-modified (AdlacZ(MM)) adenoviruses. However, in human hepatocellular Hep-G2 cells, both AdlacZ(HWGF) and AdlacZ(MM) produced significantly lower transgene expression compared with the respective control viruses. In vivo, local intravascular catheter-mediated gene transfer of a HWGF-targeted TIMP-1-encoding adenovirus (AdTIMP-1(HWGF)) significantly reduced intimal thickening in a rabbit aortic balloon denudation model (P < 0.05) compared with the control adenovirus. X-Gal staining and biodistribution analyses with TaqMan RT-PCR revealed that the cyclic peptides altered vector tropism and, in particular, reduced transduction of the liver. We found that the HWGF peptide modification increased transduction efficiency of the adenovirus-mediated gene transfer in smooth muscle cells and endothelial cells in in vitro and enhanced gene transfer to the arterial wall in vivo; that peptide modification of adenoviruses beneficially modulated tissue tropism in vivo; and that efficient TIMP-1 gene transfer reduced intimal thickening in an established restenosis model in rabbits.

Molecular Characterization of Salmonella spp. Isolated from Bulk Tank Milk and Cull Dairy Cow Fecal Samples

The consumption of meat from cull dairy cows and of raw milk has been associated with foodborne salmonellosis. This survey was conducted to establish the prevalence of Salmonella in cull dairy cow fecal samples and bulk tank milk and to determine the proportion of Salmonella-positive dairy farms (n = 30) in east Tennessee. Food and Drug Administration bacteriological analytical protocols were generally used for Salmonella isolation. Primary enrichment was performed with lactose broth, and secondary enrichment was conducted with tetrathionate broth. Eosin methylene blue, hektoen enteric, xylose lysine desoxycholate, bismuth sulfite, and brilliant green (BG) were used as isolation agars. BG agars supplemented with individual antibiotics and/or sulfur compounds were also evaluated. Six of 268 (2.24%) bulk tank milk samples and 9 of 415 (2.17%) fecal samples from 7 of 30 (25.3%) dairy farms were Salmonella-positive. Most isolates (11 of 15) were obtained between September and December. Salmonella isolates were further characterized using polyvalent somatic O Salmonella antiserum, o-nitrophenyl-β-d-galactopyranoside (ONPG), and Analytical Profile Index (API) 20E strips for Enterobacteriaceae. Serological evaluation of presumptive positive Salmonella isolates resulted in substantial numbers of false positives (41.2%). ONPG and API 20E tests enabled further biochemical distinction of the majority of Salmonella spp. from Salmonella Arizonae and closely related members of Enterobacteriaceae like Citrobacter youngae. Pulsed-field gel electrophoresis of SpeI-digested Salmonella DNA was used to subtype isolates. The isolates grouped into four clusters. The baseline information generated in this survey is being used to develop preharvest pathogen reduction programs on selected farms.

Construction of transcriptional and translational lacZ gene reporter plasmids for use in Streptococcus mutans

Reporter genes have become standard genetic tools used to evaluate either the transcriptional or the translational activity associated with genes of interest, whose products cannot be easily assayed. The lacZ gene from Escherichia coli has been used very effectively to quantify such regulated activities in many different organisms. This report describes the construction of a pair of plasmids that may be used for either transcriptional or translational lacZ gene fusions in Streptococcus mutans. The translational E. coli β-galactosidase gene ( lacZ) fusion plasmid, pALH109, as well as the transcriptional lacZ gene fusion plasmid, pALH122, have been used successfully in S. mutans to measure the activity of various PTS genes. Both plasmids employ fusions with the E. coli lacZ gene that can be easily quantified using standard O-nitrophenyl-β- d-galactopyranoside (ONPG) based enzyme assays or the more sensitive fluorometric assays using 4-methyl-umbelliferyl β- d-galactopyranoside (MUG) as the enzyme substrate. Currently, there has been only one other report of the use of lacZ as a gene reporter in S. mutans. The plasmids described in this paper will provide new tools and techniques for the analysis of S. mutans gene regulation. In addition, we have compiled the complete nucleotide sequences of these gene reporter plasmids.

Purification and some characteristics of a recombinant dimeric rhizobium meliloti β-galactosidase expressed in escherichia coli

A recombinant Rhizobium meliloti β-galactosidase was purified to homogeneity from an Escherichia coli expression system. The gene for the enzyme was cloned into a pKK223-3 plasmid which was then used to transform E. coli JM109 cells. The enzyme was purified 35-fold with a yield of 34% by a combination of DEAE-cellulose (pH 8.0) and two sequential Mono Q steps (at pH 8.0 and 6.0, respectively). The purified enzyme had an apparent molecular mass of 174 kDa and a subunit molecular weight of 88 kDa, indicating that it is a dimer. It was active with both synthetic substrates p-nitrophenyl β-D-galactopyranoside (PNPG) and o-nitrophenyl β-D-galactopyranoside (ONPG) with K m PNPG and K m ONPG of 1 mM at 25°C. The k cat/ K m ratios for both substrates were approximately 70 mM −1 sec −1, indicating no clear preference for either PNPG or ONPG, unlike E. coli β-galactosidase. After non-denaturing electrophoresis, active β-galactosidase bands were identified using 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) or 6-bromo-2-naphthyl β-D-galactopyranoside (BNG) and diazo blue B.

Disruption of Lactobacillus delbrueckii ssp. bulgaricus 11842 cells for lactose hydrolysis in dairy products: a comparison of sonication, high-pressure homogenization and bead milling

Cultures of Lactobacillus delbrueckii ssp. bulgaricus 11842 grown on whey supplemented with yeast extract were subjected to treatments using sonication, a high-speed bead mill, and a high-pressure homogenizer. The various means of disruption were compared by the release of intracellular β-galactosidase and by scanning electron microscopy (SEM). The β-galactosidase activity was measured using o-nitrophenyl-β- d-galactopyranoside (ONPG). In general, the release of active β-galactosidase was not affected by cell concentrations in the range of 12–46% (wet wt.). The maximum activity was observed after 2–3 min of bead milling and after three passages through the high-pressure homogenizer (135 MPa). The amount of active β-galactosidase released by homogenization after one passage at 200 MPa was comparable to that released between two or three passages at 135 MPa. Sonication was not as effective as the bead mill or the high-pressure homogenizer with respect to the release of β-galactosidase or the break-up of cells as evidenced by SEM and TEM. Both bead milling and high-pressure homogenization appear to be suitable for the large-scale disruption and release of β-galactosidase from L. delbruekii ssp. bulgaricus 11842.

Cloning of maltase gene from a methylotrophic yeast, Hansenulapolymorpha

The Hansenulapolymorpha maltase structural gene (HPMAL1) was isolated from a genomic library by hybridization of the library clones with maltase-specific gene probe. An open reading frame of 1695 nt encoding a 564 amino-acid protein with calculated molecular weight of 65.3 kD was characterized in the genomic DNA insert of the plasmid p51. The protein sequence deduced from the HPMAL1 exhibited 58 and 47% identity with maltases from Candidaalbicans and Saccharomycescarlsbergesis encoded by CAMAL2 and MAL62, respectively, and 44% identity with oligo-α-1,6-glucosidase from Bacilluscereus. The recombinant Hansenulapolymorpha maltase produced in Escherichiacoli hydrolyzed p-nitrophenyl-α-d-glucopyranoside (PNPG), sucrose, maltose and α-methylglucoside and did not act on melibiose, cellobiose, trehalose and o-nitrophenyl-β-d-galactopyranoside (ONPG). The affinity of the recombinant enzyme for its substrates increased in the order maltose <α-methylglucoside <sucrose <PNPG. Southern analysis revealed presence of a single maltase gene in H.polymorpha. This is the first report on the maltase gene sequence from a methylotrophic yeast.

Cationic polymer-mediated genetic transduction into cultured human chondrosarcoma-derived HCS-2/8 cells

The usefulness of three types of cationic polymer, i.e., degraded polyamidoamine (PAMAM) dendrimer (SuperFect Transfection Reagent; Qiagen), linear polyethylenimine (PEI; ExGen 500; Euromedex), and branched PEI in gene delivery into chondrocytes was examined comparatively. A plasmid vector containing the Escherichia coli LacZ (pSES.β) was combined with one of the three cationic polymers at various molar ratios and the resultant complex (polyplex) was used to transduce a human chondrocyte-like cell line, HCS-2/8. Gene expression was evaluated by an O-nitrophenyl β-d-galactopyranoside (ONPG) assay and by staining with 0.05% 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal; Nacalai Tesque). The ONPG assay showed that the highest delivery rate was achieved when 2μg of pSES.β was combined with either 21μg of dendrimer, 1.7μg of linear PEI, or 2.0μg of branched PEI. At the same DNA/polymer ratios, the proportions of X-gal-stained cells were also the highest (31.3±7.5%, 30.3±9.0%, and 8.3±3.1%, respectively). LacZ expression reached the highest level 3 days after the dendrimer-mediated transduction, and gradually declined, returning to the background level on day 14. Possible cytotoxicity was examined by trypan blue staining and phase contrast microscopic observations. Neither cytotoxicity nor morphological change was induced at the optimal dose of each polymer. The cationic polymers, particularly the degraded dendrimer and linear PEI, would be a useful nonviral vector for gene delivery to cells of chondrocytes.

Evaluation of β-Galactosidase Activity in Tissue in the Presence of Blood

The reporter gene for β-galactosidase is frequently used to determine the efficiency of gene transfer in arteries. However, blood is often present in arterial explants and may compromise the results by the presence of hemoglobin. The light absorption of hemoglobin is similar to the absorption of several colorimetric products of the commonly used β-galactosidase substrates, including o-nitrophenyl-β-D-galactopyranoside (ONPG) and chlorophenol red galactopyranoside (CPRG). This may result in false-positive measurements of β-galactosidase enzyme activity. The aim of this investigation was to determine the most appropriate method for quantification of β-galactosidase activity in the presence of blood. Colorimetric substrates (ONPG, CPRG) or the chemiluminescent Galacton-Plus substrate were used, and light absorption was measured at different concentrations of erythrocyte extract. Among the β-galactosidase substrates tested, CPRG was the most appropriate, allowing detection of enzyme activity at concentrations as low as 0.05 mU, independent of blood contamination. Addition of reducer stabilized enzyme activity for at least 5 h. Endogenous β-galactosidase activity was evaluated and used to correct results. CPRG substrate, in combination with the reducer agent mercaptoethanol, was found to be the optimal reagent for quantifying β-galactosidase activity in the presence of blood after nonviral in vivo reporter gene transfection, even with a relatively low transfer efficiency.

Functional interaction between pleiotropic transactivator pUL69 of human cytomegalovirus and the human homolog of yeast chromatin regulatory protein SPT6.

The phosphoprotein pUL69 of human cytomegalovirus (HCMV), which is a herpesvirus of considerable medical importance in immunosuppressed patients and newborns, has previously been identified as an early-late viral protein that can stimulate several viral and cellular promoters and thus exerts a rather broad activation pattern. To gain insight into the mechanism of this transactivation process, we looked for cellular factors interacting with pUL69 in a yeast two-hybrid screen. Using a B-lymphocyte cDNA library fused to the GAL4 activation domain, we identified 34 clones, 11 of which comprised one distinct gene. Interaction with this gene turned out to be very strong, producing beta-galactosidase levels 100-fold greater than the background as measured in an ONPG (o-nitrophenyl-beta-D-galactopyranoside) assay. Sequencing identified this gene as the human homolog of the yeast factor SPT6, which is thought to be involved in the regulation of chromatin structure. A direct interaction of pUL69 and the carboxy terminus of hSPT6 could be demonstrated using in vitro pull-down experiments. After having generated a specific antiserum that is able to detect the endogenous hSPT6 protein, we were able to observe an in vivo interaction of both proteins by coimmunoprecipitation analysis. The interaction domain within pUL69 was mapped to a central domain of this viral protein that is conserved within the homologous proteins of other herpesviruses such as the ICP27 protein of herpes simplex virus. Internal deletions within this central domain, as well as a single amino acid exchange at position C495, resulted in a loss of interaction. This correlated with a loss of the transactivation potential of the respective mutants, suggesting that the hSPT6 interaction of pUL69 is essential for stimulating gene expression. Furthermore, we demonstrate that the carboxy terminus of hSPT6 also binds to histon H3 and that this interaction can be antagonized by pUL69. This allows the deduction of a model by which pUL69 acts as an antirepressor by competing for binding of histones to hSPT6, thereby antagonizing the chromatin remodeling function of this cellular protein.

Development of spectrophotometric method for monitoring trans-sialidase reaction and its application

A spectrophotometric method that can monitor the trans-sialidase catalyzed reaction was devised by using a chromogenic substrate, o-nitrophenyl-β-d-galactopyranoside (ONPG), as a sialic acid acceptor. This method yielded results that were consistent with those from HPLC, and could be very conveniently used in the screening of sialic acid donor or inhibitor of trans-sialidase.

Separation and characterization of three β-galactosidases from Bacillus circulans

Crude preparation of Bacillus circulans β-galactosidase is known to have a good transglycolytic activity. Two isoforms of the enzyme have been described so far in the literature. Aiming at separating these two forms to assess their relative contribution to the regioselectivity of transglycosylation, we observed the presence of a third isoform never described before. This paper deals with the isolation procedures of the three enzymes and a re-consideration of their properties. The estimated molecular weight for the isoforms were 212 kDa (I), 145 kDa (II) and 86 kDa (III), respectively. Kinetic parameters were determined towards the hydrolysis of o-nitrophenyl-β- d-galactopyranoside (ONPG) and lactose. For ONPG the following values of K m were found: 3.6, 5.0 and 3.3 mM for I, II and III, respectively, whereas for lactose the values were 3.7, 2.94 and 2.71 mM, respectively.

RSF1010-based shuttle vectors for cloning and expression in Pasteurella multocida

The broad host-range cloning vectors, pJRD215 and pMMB67EH, were evaluated for stability and cloning efficiency in Pasteurella multocida. Transformation of P. multocida by electroporation was unreliable and poorly efficient regardless of whether the transforming DNA was isolated from E. coli or P. multocida. Both vectors contain a mob site that enabled transfer by conjugation from E. coli to P. multocida with high efficiency. Kanamycin, streptomycin, and ampicillin resistance encoded by the vectors were expressed in P. multocida. LacZ was cloned in pMMB67EH, an expression vector, and was transferred to P. multocida by conjugation. The transconjugants expressed a functional β-galactosidase as determined by o-nitrophenyl-β- d-galactopyranoside (ONPG) test. We propose the use of these cosmid and expression vectors as a shuttle vectors for cloning in P. multocida.

Inhibition of Escherichia coli β-galactosidase by 2-nitro-1-(4,5-dimethoxy-2-nitrophenyl)ethyl, a photoreversible thiol label

1-Nitro-2-phenylethene (β-nitrostyrene, 1), which is a thiol-protecting reagent (Jung, G., Fouad, H. and Heusel, G. (1975) Angew. Chem. Int. Ed. Engl. 14, 817–818), was demonstrated in this work to be an irreversible inhibitor of β-galactosidase (EC 3.2.1.23), an enzyme known to be inhibited by some thiol reagents or though modifying a methionine residue at the active site. No reversal of the inhibition was observed upon subsequent incubation with mercaptoethanol or irradiation (350 nm). 1-(4,5-dimethoxy-2-nitrophenyl)-2-Nitroethene ( 2) was also shown to be an irreversible inhibitor (94% inhibition, pH 8.3) of the enzyme. K cat values of β-galactosidase at pH 8.3 with o-nitrophenyl β- d-galactopyranoside (ONPG) as the substrate and at the highest inhibitor concentrations employed for compound 1 (4.06 · 10 −4 M) ranged from 1.67 · 10 4 s −1 after 30 min of preincubation to < 0.07 · 10 4 s −1 after 180 min preincubation. For compound 2 (9.5 · 10 −5 M) K cat values ranged from 2.70 · 10 4 s −1 following 30 min preincubation to 1.15 · 10 4 s −1 after 180 min of preincubation; the changes in K m app, however, were small. The activity was not recovered following incubation with mercaptoethanol. Since compound 2 and the inhibited enzyme are 2-nitrobenzyl derivatives, they are expected to be photosensitive and indeed, irradiation of the inhibited enzyme in the presence of mercaptoethanol resulted in recovery (89%, pH 8.3) of the enzyme activity.

Spray drying as a method for preparing concentrated cultures of Lactobacillus bulgaricus

P. TEIXEIRA, H. CASTRO AND R. KIRBY. 1995. Spray drying and freeze drying as methods for concentration of Lactobacillus bulgaricus starter cultures were compared in terms of viability, lag phase until onset of pH decrease and total acid production. For the experimental conditions used, no significant differences were detected between the methods.The effect of spray drying on the cell membrane of Lactobacillus bulgaricus was studied. Five separate methods were used to study the theory that spray drying causes cell membrane damage; three relating to leakage of intracellular components from the cell into the surrounding environment (260 and 280 nm absorbing materials, potassium ions and proteins); and two relating to increased cell permeability (increased sensitivity to NaCl and increased permeability to o‐nitrophenyl‐β‐D‐galactopyranoside (ONPG). Partial loss of some cytoplasmic material from the damaged cells was observed. The dried cells also became sensitive to NaCl and permeable to ONPG. Heat shock increased the survival of exponential cells as compared to controls but did not result in normal levels found with unshocked stationary phase cells. Heat shock had no effect on stationary phase cells. Different rehydration methods and media were investigated: slow rehydration increased survival.

Principal characteristics of β-galactosidase from Leuconostoc spp.

Abstract β-Galactosidase (β-gal) and phospho-β-galactosidase (P-β-gal) from 33 strains belonging to different species of Leuconostoc have been investigated. P-β-gal was not detected, suggesting that the lactose transport system by translocation (PEPP-TS) is not functional in Leuconostoc. A great number of strains possess a high level of β-gal in the presence of glucose. Characterization of β-gal from several strains of Leuconostoc, in comparison with an industrial strain of S. thermophilus which was used as a reference, showed that the β-gal of Leuconostoc strains has a pH optimum at 7.2. The temperature optimum varied with the strain, ranging from 43 to 58 °C. The Km values of the enzymes on substrate o-nitrophenyl-β- d -galactopyranoside (ONPG) varied from 0.68 to 4.72 m m . The Leuconostoc β-gal had a lower affinity for lactose than that of S. thermophilus. Comparison of SDS-PAGE protein profiles for a Lac− mutant and its wild Lac+ parental strain allowed the apparent molecular weight of β-gal to be estimated at approximately 80 000 for Leuc. mesenteroides subsp. mesenteroides, which is different from that of the β-gal of Leuconostoc lactis.

Rapid quantitation of recombinant retroviruses.

A method for measuring the concentration of recombinant retrovirus encoding the Escherichia coli LacZ gene is described. The assay is based on the quantitative measurement of beta-galactosidase activity in extracts of cells infected with the LacZ-encoding retrovirus. LacZ-encoded beta-galactosidase activity in transduced cells is measured using the colorimetric substrate, o-nitrophenyl beta-D-galactopyranoside (ONPG), and the results are read using an ELISA plate-reader. Because the entire assay is performed in a 96-well plate, large numbers of samples are easily measured, and the assay has the advantages of rapidity, precision, and ease of data collection. The assay was used to determine the optimal concentration of polybrene, a polycation known to enhance the infectivity of retroviruses, and can be used to evaluate other factors that affect infection, as well as the optimal conditions for production of the recombinant retrovirus.