Green fluorescent protein in Saccharomyces cerevisiae: real-time studies of the GAL1 promoter.

Green fluorescent protein (GFP) was used to study the regulation of the galactose-inducible GAL1 promoter in yeast Saccharomyces cerevisiae strains. GFP was cloned into the pGAL110 vector and transformed into the yeast strains. Time course studies comparing culture fluorescence intensity and GFP concentration were conducted along with on-line monitoring of GFP expression. Our results demonstrated that GFP fluorescence could be used as a quantifiable on-line reporter gene in yeast strains. The effect of an integrated GAL10p-GAL4 transcription cassette was investigated. Induction time studies showed that there was no significant difference in GFP expression level by adding galactose at different culture times. A wide range of galactose concentrations was used to study the initial galactose concentration effect on GFP expression kinetics. A minimum of 0.05 g/L galactose doubled the GFP fluorescence signal as compared to the control, whereas 0.1 g/L gave the highest specific GFP yield. A simple analytical model was proposed to describe GFP expression kinetics based on the experimental results. In addition, this GFP-based approach was shown to have potential use for high-throughput studies. The use of GFP as a generic tool provided important insights to the GAL expression system and has great potential for further process optimization applications.

Purification of virus-like particles of recombinant human papillomavirus type 11 major capsid protein L1 from Saccharomyces cerevisiae.

Recombinant major capsid protein, L1 (M(r) = 55,000), of human papillomavirus type 11 was expressed intracellularly at high levels in a galactose-inducible Saccharomyces cerevisiae expression system by an HPV6/11 hybrid gene. The capsid protein self-assembled into virus-like particles (VLPs) and accounted for 15% of the total soluble protein. A purification process was developed that consisted of two main steps: microfiltration and cation-exchange chromatography. The purified VLPs were 98% homogeneous, and the overall purification yield was 10%. The final product was characterized by several analytical methods and was highly immunogenic in mice.

Age, geographic, and temporal distribution of fecal shedding of Cryptosporidium parvum oocysts in cow-calf herds.

OBJECTIVE: To evaluate fecal shedding of Cryptosporidium parvum from California cow-calf herds with respect to age, geographic region, temporal effects, and association with watery feces. ANIMALS: Cows and calves from 38 beef cow-calf operations. PROCEDURE: Fecal specimens were collected and examined for C parvum oocysts, using immunofluorescent microscopy. Associations between age, geographic region, month of collection, watery feces, and likelihood of shedding C parvum were evaluated. RESULTS: 3.9% of cattle were shedding C parvum oocysts. Prevalence of shedding among calves ranged from 0 to 13%, and was 0.6% among cattle > or = 12 months old. The odds of shedding C parvum among 2-month-old calves were 41 times greater than among cattle > 4 months old. The odds of shedding C parvum among cattle tested in May were 8.7 times greater than among cattle tested during June, July, or August. The odds of infected individuals having watery feces were 3 to 4 times greater than for noninfected individuals, but the etiologic fraction was only 8 to 9%. CONCLUSIONS AND CLINICAL RELEVANCE: Substantial fecal shedding of C parvum by cow-calf herds was limited to calves 1 to 4 months old, with low prevalence detected in older animals. Risk of contamination of watersheds with C parvum was limited to those periods when young calves were in the herd. Although the odds of having watery feces were greater for animals infected with C parvum than for noninfected animals, the low etiologic fraction suggests that most calves with watery feces were not infected with C parvum.

Expression and purification of recombinant tick anticoagulant peptide (Y1W/D10R) double mutant secreted by Saccharomyces cerevisiae.

A double mutant of tick anticoagulant peptide (TAP) was cloned as a chimeric fusion with the yeast alpha-mating factor pre-proleader peptide. Expression in yeast (Saccharomyces cerevisiae) resulted in the secretion of the TAP mutein into the culture medium. An HPLC-based assay was used to screen yeast strains to find those giving highest expression levels. Efficiency of cleavage at the junction of the leader-TAP mutein varied from strain to strain, and a rapid purification method followed by N-terminal sequence analysis was used to identify a host strain that minimized undesirable cleavage products. A purification scheme was developed which separated the TAP mutein from improperly processed peptides present in the medium. This scheme employed cation-exchange chromatography and reversed-phase HPLC. Scale-up of the process was successful and produced 100 mg of fully functional TAP mutein of >96% homogeneity from a 50-L yeast culture.

Human papillomavirus type 11 (HPV-11) neutralizing antibodies in the serum and genital mucosal secretions of African green monkeys immunized with HPV-11 virus-like particles expressed in yeast.

It has been shown previously that immunization of animals with recombinant virus-like particles (VLPs) consisting of the viral capsid proteins L1 or L1 plus L2 protected animals against experimental viral challenge. However, none of these experimental models addresses the issue of whether systemic immunization with VLPs elicits a neutralizing antibody response in the genital mucosa. Such a response may be necessary to protect the uterine cervix against infection with genital human papillomavirus (HPV) types. African green monkeys systemically immunized with HPV-11 VLPs expressed in Saccharomyces cerevisiae and formulated on aluminum adjuvant elicited high-titered HPV-11 VLP-specific serum antibody responses. Sera from these immunized monkeys neutralized HPV-11 in the athymic mouse xenograft system. Significant levels of HPV-11-neutralizing antibodies also were observed in cervicovaginal secretions. These findings suggest that protection against HPV infection of the uterine cervix may be possible through systemic immunization with HPV VLPs.

Adenine quantitation in yeast extracts and fermentation media and its relationship to protein expression and cell growth in adenine auxotrophs of Saccharomyces cerevisiae.

We have described a method to reliably measure the free adenine content of yeast extract powders or the adenine concentrations found in chemically-defined and complex fermentation samples. This method relies on the selective precolumn derivatization of adenine with chloroacetaldehyde to form the fluorescent adenine adduct 1,N6-ethenoadenine. The derivatized adenine can then be resolved from other components found in samples with reverse phase HPLC and selectively monitored with fluorescence. This method was then used to study the adenine nutritional requirements of adenine auxotrophs of recombinant Saccharomyces cerevisiae. The adenine content of individual yeast extract powders was examined in relation to the cell mass (dry cell weight, DCW) achieved in culture media formulated with these powders. A general increase in DCW was observed with increasing adenine concentration in the yeast extract. Conversely, we observed that as adenine concentration increased in complex media the expression levels of a heterologous protein decreased. This method also allowed us to examine the adenine/DCW ratio in both steady-state continuous culture and batch culture. In both cases, the total in vivo adenine content as measured by the amount of adenine utilized from the culture media was estimated to be ca. 25-40 mg/g DCW. However, data suggest that this value is in excess of what is strictly required for cell growth and represents the quantity of adenine required to saturate intracellular pools of adenine or adenine metabolites. A minimum requirement for cell growth is at least as low as 12.5 mg of adenine/g of cells.

Continuous culture study of the expression of hepatitis B surface antigen and its self-assembly into virus-like particles in Saccharomyces cerevisiae.

We have studied the growth rate dependence of hepatitis B surface antigen (HBsAg) p24(s) monomer and lipoprotein particle synthesis produced in Saccharomyces cerevisiae using galactose-limited continuous culture. The hepatitis B virus S gene, which encodes the p24(s) monomer, is transcribed under the control of the GAL 10p on a chimeric 2-microm plasmid harbored in a haploid yeast strain. Monomers autonomously form lipoprotein aggregates (particles) in vivo using only host-cell-derived components. Steady states were evaluated in a range from 0.015 h(-1) to washout (0.143 h(-1)). Both p24(s) monomer and HBsAg particle levels, at steady state, varied in an inverse linear manner with growth rate. A consistent excess of total p24(s) monomer to HBsAg particle, estimated at five- to tenfold by mass, was found at all dilution rates. The average copy number of the 2-microm plasmid (carrying LEU2 selection) remained constant at 200 copies per cell from washout to 0.035 h(-1). Surprisingly, the average copy number was undetectable at the lowest dilution rate tested (0.015 h(-1)), even though HBsAg expression was maximal. Total p24(s) monomer and HBsAg particle values ranged twofold over this dilution rate range. No differences in the trends for HBsAg expression and average copy number could be detected past the critical dilution rate where aerobic fermentation of galactose and ethanol overflow were observed. HBsAg expression in continuous culture was stable for at least 40 generations at 0.100 h(-1).

Sequence determination of human papillomavirus type 6a and assembly of virus-like particles in Saccharomyces cerevisiae.

Human papillomavirus 6a (HPV6a), the most abundant HPV6 subtype, was detected in a vulvar condyloma acuminatum. The complete genome of HPV6a was cloned, and its DNA sequence was shown to be over 97% identical to the HPV6b sequence. Of the eight open reading frames (ORFs) of HPV6a, only the imputed amino acid sequence of the major capsid protein L1 was identical to the corresponding HPV6b sequence; all other HPV6a ORFs showed amino acid changes compared to the HPV6b ORFs. The HPV6a L1 or the L1 + L2 ORFs were expressed in the yeast Saccharomyces cerevisiae. Self-assembly of the L1 capsid protein into virus-like particles (VLPs) was demonstrated both in the L1 as well as L1 + L2 coexpressing yeast strains. Copurification of the L1 and L2 proteins showed complex formation of the L1 and L2 proteins in the yeast-derived VLPs of coexpressing strains.

Recent advances in the large scale fermentation of Neisseria meningitidis group B for the production of an outer membrane protein complex.

The Outer Membrane Protein Complex (OMPC) of the bacterium Neisseria meningitidis group B has been used successfully as a protein carrier in a Haemophilus influenza type b (Hib) polysaccharide conjugate vaccine and a Streptococcus pneumoniae (Pn) polysaccharide conjugate vaccine to elicit antipolysaccharide immune responses in young infants. The OMPC carrier is derived by detergent extraction of whole cells and, thus, the consistent generation of suitable biomass is central to an effective production process. Therefore, we have developed a large-scale, high-cell density (5 g/L dry cell weight) fermentation process for the cultivation of N. meningitidis B11. Since current requirements for the production of human biologics mandate strict control of all aspects of the manufacturing process, several key features of the process, including a chemically defined medium and a rational event-based harvest criterion, support current good manufacturing practice (cGMP) and increased productivity.

Development of scale-down techniques for investigation of recombinant Escherichia coli fermentations: acid metabolites in shake flasks and stirred bioreactors.

We have developed shake-flask screening conditions that are predictive of specific expression of the chimeric toxin, TGF alpha-PE40, by recombinant Escherichia coli JM109 in stirred bioreactors. When a nutrient-rich stirred bioreactor medium was used in shake flasks, neither the extent of growth nor the specific level of recombinant protein expression duplicated the performance in stirred bioreactor fermentations. Incomplete oxidation of glucose and concomitant accumulation of organic acid metabolites, as well as oxygen limitation and lack of pH control, were examined as contributors to the poorer performance in the flask. The medium buffering capacity, initial glucose level, and flask aeration were evaluated to establish the limits of "scale-down" conditions for expression both in a complex nutrient medium (M101) similar to that used in stirred bioreactors and in a defined (FM) medium. Acid metabolites and ethanol were measured as indicators of carbon flow from glucose as well as indirect indicators of oxygen limitation. For the complex M101 medium, optimal shake-flask performance in 250-mL, nonbaffled flasks at 37 degrees C occurred with 0.3 x medium strength, supplementation with 0.3 m HEPES buffer (pH 7.5), and 10 mL of medium per flask. Cultures grown under these conditions produced a maximum density of 3.6 g of dry cell weight/L (as estimated by absorbance measurements at 600 nm) and maintained a pH near neutrality. Additionally, metabolite markers of anaerobic or microaerobic conditions, such as ethanol, lactate, and pyruvate, were not detected, and specific expression of TGF alpha-PE40 was comparable to stirred bioreactors induced for expression at various biomass levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Physiological effects of TGF(alpha)-PE40 expression in recombinant Escherichia coli JM109.

Physiological effects of isopropyl-thiogalactopyranoside (IPTG) induction were examined in Escherichia coli strain JM109 expressing a fusion protein composed of transforming growth factor alpha and a 40-kD portion of Pseudomonas aeruginosa exotoxin A (TGF(alpha)-PE40) under control of the tac promoter. Fermentations at the 15-L scale in complex medium compared growth and metabolite profiles of the untransformed JM109 host strain, the strain transformed with the vector lacking the TGF(alpha)-PE40 open reading frame (JM109[pKK2.7]), and the strain with the complete plasmid for TGF(alpha)-PE40 expression (JM109[pTAC-TGF57-PE40]). Metabolite and growth profiles of JM109 (pTAC-TGF57-PE40) cultures changed significantly in IPTG-induced versus uninduced cultures. Prior to induction, glucose was metabolized to acetate or completely oxidized to CO(2). Following induction, pyruvate was also excreted in addition to acetate. In the absence of inducer, pyruvate was excreted by JM109 (pTAC-TGF57-PE40) only when dissolved oxygen levels fell to less than 10% of saturation (microaerobic rather than anaerobic conditions). The untransformed JM109 host strain or JM109 (pKK2.7) did not excrete pyruvate in the presence or absence of inducer, although JM109 (pKK2.7) exhibited a pattern of growth following addition of IPTG that closely resembled JM109 (pTAC-TFG57-PE40). Fermentations of JM109 (pTAC-TFG57-PE40) in a synthetic medium supported lower expression levels, but resulted in similar alterations in metabolite profiles. Induction in synthetic medium resulted in pyruvate excretion without further acetate accumulation. Taken together, these data suggest that one consequence of TGF(alpha)-PE40 expression in JM109 is altered patterns of pyruvate oxidation.

Acidic Conditions Are Not Obligatory for Onset of Butanol Formation by Clostridium beijerinckii (Synonym, C. butylicum).

Factors that may initiate the metabolic transition for butanol production were investigated in batch cultures of Clostridium beijerinckii (synonym, Clostridium butylicum) VPI 13436. Cultures maintained at pH 6.8 produced nearly as much butanol as those incubated without pH control, indicating that neither a change in the culture pH nor acid conditions per se are always required to initiate solvent formation. Acetate and butyrate levels at the onset of butanol production were dependent on the pH at which the cultures were maintained. Cultures maintained at pH 6.8 could be accelerated into solvent production by artificially lowering the pH to 5.0 or by the addition of acetate plus butyrate without a pH change (but neither acid alone was effective). Solvent production was associated with slower rates of growth and general metabolism, and it did not show a requirement for mature spore formation. We speculate that a slowdown in metabolism, which may be brought about by several conditions, is mechanistically related to the onset of butanol production. Extracts of solvent-producing cells contained acetoacetate decarboxylase activity as well as higher NADP-linked butanol dehydrogenase and lower hydrogenase activities than extracts of acid-producing cells. Solvent production did not appear to involve an enhanced ability to catalyze H(2) oxidation.

Acetone, Isopropanol, and Butanol Production by Clostridium beijerinckii (syn. Clostridium butylicum) and Clostridium aurantibutyricum.

Thirty-four strains representing 15 species of anaerobic bacteria were screened for acetone, isopropanol, and n-butanol (solvent) production. Under our culture conditions, several strains of Clostridium beijerinckii and C. aurantibutyricum produced at least 40 mM n-butanol (C. acetobutylicum strains produced up to 41 mM n-butanol under similar conditions). Both solvent-producing and non-solvent-producing strains of C. beijerinckii have high DNA homology with a reference strain of C. beijerinckii. Strains labeled "Clostridium butylicum" are phenotypically similar to C. beijerinckii and showed at least 78% DNA homology to a reference strain of C. beijerinckii. Therefore, these "C. butylicum" strains are members of C. beijerinckii. An earlier DNA homology study has shown that C. beijerinckii, C. aurantibutyricum, and C. acetobutylicum are distinct species.

Fumarate reduction and product formation by the Reiter strain of Treponema phagedenis.

The catabolic pathways for butyrate, acetate, succinate, and ethanol formation by the Reiter strain of Treponema phagedenis were investigated. Enzyme activities were demonstrated for glucose catabolism to pyruvate by the Embden-Meyerhof-Parnas pathway. Butyrate formation from acetyl-coenzyme A (acetyl-CoA) does not generate ATP by substrate level phosphorylation and involves NAD+-dependent 3-hydroxybutyryl-CoA dehydrogenase and NAD(P)+-independent butyryl-CoA dehydrogenase activities. Butyrate is formed from butyryl-CoA in a CoA transphorase reaction. Phosphate acetyltransferase and acetate kinase activities convert acetyl-CoA to acetate. An NADP+-dependent alcohol dehydrogenase participates in ethanol formation; however, the manner in which acetyl-CoA is reduced to acetaldehyde is unclear. A membrane-associated fumarate reductase was found which utilized reduced ferredoxin or flavin nucleotides as physiological electron donors. Additional electron carriers may also be involved in electron transfer for fumarate reduction. Strains of Treponema denticola, T. vincentii, and T. minutum utilized fumarate without succinate formation, whereas strains of T. phagedenis and T. refringens formed succinate from exogenously supplied fumarate.

Pyruvate oxidation by the Reiter strain of Treponema phagedenis.

Spectrophotometric assays of pyruvate oxidation catalyzed by extracts of the Reiter strain of Treponema phagedenis indicated that viologen dyes, flavin nucleotides, and a ferric iron chelate, but not pyridine nucleotides, were utilized as electron acceptors. Benzyl viologen-linked activity partially sedimented during ultracentrifugation and appeared similar to clostridial pyruvate:ferredoxin oxidoreductase with respect to the spectral properties of the enzyme chromophore. Electron carrier activity in treponemal extracts was quantitated by a metronidazole-linked assay in which the oxidation of pyruvate by carrier-depleted extracts led to the reduction of electron carrier in the crude extracts which then reduced metronidazole. The rate of metronidazole reduction was proportional to the amount of electron carrier present in the assay. Electron carrier activity in Triton X-100-solubilized, crude extracts partially purified by DEAE-cellulose chromatography and gel filtration was attributed to a protein possessing the spectral and physical properties of a ferredoxin. A similar protein appeared to be present in extracts of Treponema denticola ST10.

Lipid catabolism of cultivated treponemes.

Treponema require long-chain fatty acids for growth in vitro. Serum, added to culture media, provides a source of long-chain fatty acids. These fatty acids, however, are esterified to triglycerides, phospholipids, and cholesterol. In this study, the major pathways of complex lipid catabolism in T. phagedenis, T. denticola, T. refringens, T. minutum, and T. vincentii were investigated. Lipase activity was demonstrated in five Treponema species using four lipid substrates. Chromatographic data demonstrated that, during growth, treponemes completely utilized lysophosphatidylcholine, present in serum-supplemented culture media, while phosphatidylcholine and phosphatidylinositol were not utilized. Phospholipase B and glycerophosphorylcholine diesterase activities were demonstrated in the five species of Treponema studied. Treponema phagedenis and T. denticola had phosphatase activity, while T. refringens, T. minutum, and T. vincentii did not have an acid phosphatase activity. Phospholipase A, C, and D and alkaline phosphatase activities were not found in five species of Treponema. Based on the enzymes demonstrated in this study, two pathways of phospholipid catabolism are proposed.

Studies of the microaerophilic nature of Campylobacter fetus subsp. jejuni. II. Role of exogenous superoxide anions and hydrogen peroxide.

The addition of bovine superoxide dismutase to Brucella broth or Brucellar agar greatly echanced the oxygen tolerance of Campylobacter fetus subsp. jejuni strain H840 (ATCC 29428). Catalase also enhanced oxygen tolerance, but to a lesser extent. These enzymes must act externally to the bacteria. All of the diverse compounds which enhance oxygen tolerance of C. fetus, including nor-epinephrine and a combination of ferrous sulfate, sodium metabisulfite, and sodium pyruvate, share the ability to quench either superoxide anions or hydrogen peroxide. On the basis of these and other data, we propose that C. fetus is more sensitive to exogenous superoxide anions and hydrogen peroxide than are aerotolerant bacteria, despite the occurrence of superoxide dismutase and catalse activities in C. fetus. Compounds that enhance oxygen tolerance in C. fetus appear to act by quenching superoxide anions and hydrogen peroxide which occur spontaneously in the culture medium.

Improved media for growth and aerotolerance of Campylobacter fetus.

The microaerophilic nature of Campylobacter fetus has complicated its recovery from human and animal sources. In this study, modifications of brucella agar and broth were tested for enhancement of growth and aerotolerance of 64 strains of C. fetus, representing each subspecies. Brucella agar supplemented with 0.025% each FeSO4 7H2O, sodium metabisulfite, and sodium pyruvate, supported growth of 98, 77, and 63% of the strains at 6% O2, 17% O2, and 21% O2, respectively. Unsupplemented brucella agar supported growth of 94, 48, and 20% of the strains. Brucella broth supplemented with 0.2% FeSO4.7H2O, 0.025% sodium metabisulfite, and 0.05% sodium pyruvate supported growth of 98% of the strains at 21% O2, compared to 75% with unsupplemented brucella broth. With both the supplemented agar and broth, growth responses occurred 1 to 2 days earlier than usual. Growth and aerotolerance of three strains of Campylobacter sputorum subsp. bubulus were not enhanced by the supplements.