Total Soluble Cell Protein Research Articles

Construction of viral-based expression vectors for high-level production of human interferon alpha 2b in plants

Human interferon (hINF) alpha 2b is clinically important pharmaceutical product included in combinatory therapy against chronic hepatitis C and B and complex therapy against several cancer diseases. Here, we created the genetic constructions, based on genome elements of potato virus X (PVX), carrying the infα2b gene for transient expression in plant cells. The created plasmid vector constructions were tested through Agrobacterium-mediated transient gene expression method in two plant species—Nicotiana benthamiana and Ocimum basilicum (sweet basil). Production of recombinant hINF alpha 2b was more efficient in N. benthamiana than that in O. basilicum plants. The average yield of hINF alpha 2b produced in N. benthamiana plants was 0.56 mg/g of fresh leaf weight (FW) or 6% of the total soluble cell proteins (TSP). The maximal level reached up to 1.2 mg/g FW or 9% TSP. We estimated that about 0.67 mg of hINF can be obtained from one N. benthamiana plant. The yield of hINF alpha 2b obtained with the PVX-based expression cassette was about 80 times higher than the yield of hINF alpha 2b obtained with a simple expression cassette in which the infα2b gene was controlled by the 35S promoter of cauliflower mosaic virus.Key points• PVX-based expression vectors provide efficient transient expression of infα2b gene• N. benthamiana plants can produce human interferon alpha 2b at high levels• The yield of the hINF α2b reached up to 1.2 mg/g of fresh leaf weight

Identification of Lactobacillus spp. Isolated from Different Phases During the Production of a South African Fortified Wine

Fortified wines contain a high level of unfermented sugars and are prone to spoilage by alcohol-tolerant lactic acid bacteria. A total of 62 strains were isolated from various production stages of one of the more popular fortified wines produced in South Africa. The strains were identified by using numerical analysis of total soluble cell protein patterns and 16S rRNA sequence analyses. The species most frequently isolated were Lactobacillus vermiforme (24 strains) and Lactobacillus casei subsp. casei (32 strains). Twenty-four of the strains of L. vermiforme, three strains of Lactobacillus buchneri, one strain of Lactobacillus plantarum and two strains of L. casei subsp. casei were isolated from spoiled fortified wine which contained 22% (vol/vol) ethanol. The majority of strains of L. casei subsp. casei (25 of the 32) and two strains of Lactobacillus zeae were isolated from wine before submerged fermentation. Five strains of L. casei subsp. casei were isolated from wine undergoing submerged fermentation, with an alcohol content of 11.92% (vol/vol). No strain was isolated from unbottled wine which underwent the complete fermentation process and with an alcohol content of 17.20% (vol/vol). Three distinct phenotypic groups of L. vermiforme were identified at r ≥ 0. 70, separate from Lactobacillus brevis,' L. buchneri and Lactobacillus hilgardii. Three phenotypic clusters have been identified for L. casei subsp. casei. This is the first report of the presence of L. vermiforme, L. zeae, L. casei subsp. casei and L. plantarum in fortified wines.

Characterization of Killer Yeast Isolates from Chenin blanc Grapes and Grape Skins

Wild-type killer yeast strains isolated from six South African wineries were identified using classical taxonomic methods. They were further characterized according to their cross-reactions with reference killer yeasts (K1-Kn) and by electrophoresis of their double-stranded RNA molecules. All isolates belonged to the K2 phenotype and were identified as strains of Saccharomyces cerevisiae and Saccharomyces bayanus. The killer strains differed substantially in their ability to kill a sensitive wine yeast (Geisenheim GS-1). This phenomenon may be attributed to strain differences among the killer yeasts as was shown by electrophoresis of total soluble cell proteins and gas chromatographic analysis of cellular fatty acids.

Killer Yeasts - Cause of Stuck Fermentations in a Wine Cellar

Sluggish fermentations in five fermenters in a wine cellar were investigated. Methylene blue-stains of yeast suspensions revealed that approximately 90% of the total yeast population in each of the fermenters were dead. The viable cells in each fermenter were killer yeasts. Polyacrylamide gel electrophoresis of total soluble cell proteins showed that the same killer yeast occurred in each of the five fermenters. The effect of killer yeast on viability and fermentation activity of the wine yeast was studied in an enriched grape juice medium at 20°C and 30°C. Death rate of the wine yeast was considerably higher in the presence of the killer yeast and fermentations were retarded at both temperatures. The killer yeast induced flocculation of the non-flocculent wine yeast.

Biochemical, physiological and molecular characteristics of chickpea rhizobacteria from Kurdistan province, Iran

Kurdistan province of Iran is one of the main places for producing chickpea, and there is no published research on root-nodulating bacteria of this crop. Plant samples were collected and a total of 73 Rhizobium strains were isolated from root nodules. Nodulation test was done on chickpea plants. Phenotypic characteristics of the 16 representative strains were determined based on the standard bacteriological methods. Total soluble cell protein patterns by electrophoresis approach (SDS-PAGE) showed heterogeneity among the tested rhizobia strains. Based on the phenotypic features, Rhizobium strains of three groups belong to different species of the genus Mesorhizobioum including M. ciceri and M. mediterraneum and Mesorhizobium sp. The PCR technique was employed for amplification of 16S rDNA and atpD genes. For further characterisation, amplified fragment of 16S rDNA gene from a representative strain (AK21) using primers 41F and 1488R was subjected to sequencing. Sequences were aligned by BLAST software at NCBI GenBank and results showed 99% similarity with M. mediterraneum strain BKBCF3q.

Fermentation process for tetrameric human collagen prolyl 4-hydroxylase in Escherichia coli: Improvement by gene optimisation of the PDI/β subunit and repeated addition of the inducer anhydrotetracycline

The collagen prolyl 4-hydroxylases (C-P4Hs) that reside within the lumen of the endoplasmic reticulum (ER) are the key enzymes in the biosynthesis of collagens. The vertebrate enzymes are alpha(2)beta(2) tetramers consisting of two catalytic alpha subunits and two beta subunits that are identical to protein disulfide isomerase (PDI). Cytoplasmic production of an active human C-P4H has recently been described in the Origami (trxB gor) mutant Escherichia coli using a bicistronic vector with independent control of the alpha and PDI/beta subunit expression by the tetA and T5-lac promoters, respectively, enabling sequential induction (Neubauer, A., Neubauer, P., Myllyharju, J., 2005. High-level production of human collagen prolyl 4-hydroxylase in Escherichia coli. Matrix Biol. 24, 59-68). We show here that the yield of active C-P4H in shake flasks is increased 50-fold by improving the expression level of the PDI/beta subunit through gene optimisation. We also found that stable expression of the alpha subunit mRNA in a fed-batch fermentation process requires repeated additions of anhydrotetracycline. This finding may be of a wider general importance for the use of the tetA promoter in fed-batch cultivations, especially if recombinant proteins are expressed during long production phases. We also show that growth of the E. coli Origami strain to high cell density on a complex medium with consecutive sequential induction is difficult to achieve and that optimisation of similarly complicated systems can greatly benefit from the use of quantitative mRNA analysis for the evaluation of transcriptional bottlenecks. The optimisation approach resulted in a fermentation yield of 143 mg L(-1) of active C-P4H, corresponding to approximately 7.5% of the total soluble cell protein.

Species attribution and distinguishing strains of Oenococcus oeni isolated from Chinese wines

Twenty-four strains of Oenococcus oeni were isolated from different Chinese wines. Differentiation of isolates was carried out by analysis of total soluble cell protein patterns and random amplified polymorphic DNA (RAPD) patterns. The results indicated that the total soluble cell protein patterns could be used to distinguish different genera but fail to distinguish different strains. It was also found that strain RAPD pattern can successfully distinguish isolates by UPGMA analysis. The RAPD profiles (107 different prints) were strain specific and two main groups of strains were screened.

Identification of lactic acid bacteria isolated from South African brandy base wines

In brandy base wines, no sulphur dioxide is used and it therefore is ideal for the proliferation of lactic acid bacteria. As part of an extensive taxonomic survey within the ecological framework of South African vineyards and wineries, and the influence of naturally occurring lactic acid bacteria on the quality of wine and brandy, a total of 54 strains were isolated from grape juice and at different stages of brandy base wine production. The strains were identified using numerical analysis of total soluble cell protein patterns, 16S rRNA sequence analyses and polymerase chain reaction (PCR) using species-specific primers. The predominant species was Oenococcus oeni (22 strains), but Lactobacillus brevis (8 strains), Lactobacillus paracasei (8 strains) and Lactobacillus plantarum (6 strains) were also isolated frequently. Many of the O. oeni strains were isolated from brandy base wines after completion of spontaneous malolactic fermentation (MLF). The Lactobacillus spp. were isolated from all the different stages of brandy base wine production. Lb. plantarum was the dominant species in the juice, but disappeared during the later stages of production. However, Lactobacillus hilgardii, Lb. brevis and Lb. paracasei were also isolated from base wine after spontaneous MLF. Strains identified as Lactobacillus vermiforme were isolated during the alcoholic fermentation and after MLF have been completed. Total soluble cell protein patterns grouped O. oeni strains into two phenotypic groups. Two phenotypic clusters have also been identified for the Lb. brevis isolates . The Lb. paracasei isolates all grouped in one cluster. This is the first report of the presence of Lb. paracasei and Lb. vermiforme in brandy base wines. The presence of the Lactobacillus spp. could be correlated to the decrease in quality of the base wine and distillate, while O. oeni strains were found to have a more favourable influence on the quality of base wine and distillates. These results shed some light on the ecology and oenological influence of lactic acid bacteria (LAB) on the quality of South African brandy.

Identification of heterofermentative lactobacilli isolated from pig faeces by numerical analysis of total soluble cell protein patterns and RAPD-PCR.

The study deals with a number of heterofermentative Lactobacillus strains isolated from pig faeces and their identification. SDS-PAGE of total soluble cell proteins and RAPD-PCR profiles were used to identify the strains isolated from pig faeces. Protein profiles obtained with SDS-PAGE revealed that 15 strains clustered at r >or= 0.78 with Lactobacillus buchneri and nine strains at r >or= 0.77 with two reference strains of Lactobacillus reuteri. The identity of the strains was confirmed with RAPD-PCR. Numerical analysis of protein profiles and RAPD-PCR proved valuable in the differentiation of Lactobacillus spp. isolated from pig faeces. This is the first report on the association of Lact. buchneri with pig faeces.

Identification of lactic acid bacteria isolated from human vaginal secretions.

A total of 57 lactic acid bacteria were isolated from the vaginal secretions of 259 patients. Of these strains, 37 were isolated from patients attending pre-natal clinics and the remaining strains from patients attending post-natal clinics. The strains were identified by using simple physiological and biochemical tests and their phenotypic relatedness determined by numerical analysis of total soluble cell protein patterns. The genotypic relatedness of representative strains selected from each of the protein profile clusters was determined by numerical analysis of the DNA banding patterns obtained from RAPD-PCR. The majority of lactobacilli isolated belonged to the species Lactobacillus pentosus, Lactobacillus fermentum and Enterococcus faecium. A few strains of Lactobacillus plantarum and Weissella viridescens were also isolated. One strain, TV 1029, grouped into the same protein profile cluster as E. faecium, but revealed a DNA banding pattern closer related to Enterococcus faecalis. This is the first report of W. viridescens associated with the human vagina.

Cloning, overexpression, and purification of functional human purine nucleoside phosphorylase

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for human PNP causes T-cell deficiency as the major physiological defect. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant tissue rejection, psoriasis, rheumatoid arthritis, lupus, and T-cell lymphomas. Human PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation. In addition, bacterial PNP has been used as reactant in a fast and sensitive spectrophotometric method that allows both quantitation of inorganic phosphate (P i) and continuous assay of reactions that generate P i such as those catalyzed by ATPases and GTPases. Human PNP may therefore be an important biotechnological tool for P i detection. However, low expression of human PNP in bacterial hosts, protein purification protocols involving many steps, and low protein yields represent technical obstacles to be overcome if human PNP is to be used in either high-throughput drug screening or as a reagent in an affordable P i detection method. Here, we describe PCR amplification of human PNP from a liver cDNA library, cloning, expression in Escherichia coli host, purification, and activity measurement of homogeneous enzyme. Human PNP represented approximately 42% of total soluble cell proteins with no induction being necessary to express the target protein. Enzyme activity measurements demonstrated a 707-fold increase in specific activity of cloned human PNP as compared to control. Purification of cloned human PNP was achieved by a two-step purification protocol, yielding 48 mg homogeneous enzyme from 1 L cell culture, with a specific activity value of 80 U mg −1.

Gene cloning, overexpression and biochemical characterization of the peptide amidase from Stenotrophomonas maltophilia.

The peptide amidase (Pam) from the gram-negative bacterium Stenotrophomonas maltophilia catalyzes predominantly the hydrolysis of the C-terminal amide bond in peptide amides. Its gene ( pam) was isolated by Southern hybridization using a DNA probe derived from the known N-terminal amino acid sequence. Pam is a member of the amidase signature family and was identified as a periplasmic protein by an N-terminal signal peptide found in the gene. The processed protein consists of 503 amino acids with a molecular mass of 53.5 kDa. The recombinant enzyme with a C-terminal His(6) tag has a monomeric structure and its isoelectric point is 6.3. The dipeptide amide L-Ala- L-Phe-NH(2) is hydrolyzed in the absence of cofactors to L-Ala- L-Phe-OH and ammonia with V(max)=194 U/mg and K(m) <0.5 mM. The natural function of Pam remains unclear. Chymostatin ( K(i)<0.3 microM) and Pefabloc SC ( K(i) not determined) were identified as inhibitors. When the gene was expressed in Escherichia coli on a 12-l scale, the specific activity in the crude extract was 60 U/mg, compared to 0.24 U/mg in S. maltophilia. In the expression system, Pam made up about 31% of the total soluble cell protein. From 75 g wet cells, 2.1 g of >95% pure enzyme was obtained, which corresponds to a total activity of 416,000 units.

Isolation, identification and changes in the composition of lactic acid bacteria during the malting of two different barley cultivars

Malt has a complex microbial population, which changes as the malting process commences. Little is known about the proliferation of lactic acid bacteria (LAB) in each of the malting phases. In this study, we determined the number of LAB present in the different phases of malting with Clipper and Prisma barley cultivars. The strains were identified to species level by using numerical analysis of total soluble cell protein patterns, RAPD–PCR banding patterns and 16S rRNA sequencing. The number of viable LAB in the barley before steep was higher in Prisma than Clipper (7.6×10 4 and 1.2×10 3 cfu/g, respectively). Despite this, the number of viable cells recorded in the first steep water was slightly higher for Clipper (9.0×10 5 cfu/g) than Prisma (5.5×10 5 cfu/g). More or less, the same cell numbers were recorded for the two barley cultivars after the first and second dry stands. Both cultivars displayed more or less the same cell numbers (3.7×10 7 for Clipper and 3.2×10 7 cfu/g for Prisma) after the third day of germination. However, a higher number of LAB were detected in the kilned Prisma malt (6.9×10 4 cfu/g) than the Clipper malt (1.5×10 4 cfu/g). Leuconostoc argentinum, Le. lactis and Weissella confusa were the most predominant in both cultivars. A few strains were identified as W. paramesenteroides (four strains), Lactobacillus casei (five strains), Lactococcus lactis (five strains) and Lb. rhamnosus (two strains). Lb. casei and Lb. rhamnosus were not isolated from the Prisma cultivar, while W. paramesenteroides and Lc. lactis were absent in the Clipper cultivar. Kilned malt of the Clipper cultivar contained predominantly Le. argentinum, whereas the Prisma cultivar contained mainly Lc. lactis. To our knowledge, this is the first report of LAB in Clipper and Prisma barley and the various malting phases. The influence of the various groups of lactic acid bacteria on the fermenting ability of brewers' yeast is currently being determined.

Cloning and overexpression in soluble form of functional shikimate kinase and 5-enolpyruvylshikimate 3-phosphate synthase enzymes from Mycobacterium tuberculosis.

Tuberculosis (TB) resurged in the late 1980s and an estimated 1.87 million people died of TB in 1997. The reemergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons, and the proliferation of multidrug-resistant strains have created a need to develop new antimycobacterial agents. The existence of a shikimate pathway has been predicted by the determination of the genome sequence of Mycobacterium tuberculosis. The M. tuberculosis aroK-encoded shikimate kinase and aroA-encoded 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase were cloned and the enzymes overexpressed in soluble form. Overexpression was achieved without isopropyl beta-d-thiogalactoside induction, and cells grown to stationary phase yielded approximately 30% of target proteins to total soluble cell proteins. Enzyme activity measurements using coupled assays demonstrated that there was a 328-fold increase in specific activity for shikimate kinase and 101-fold increase for EPSP synthase.

Taxonomy of obligately homofermentative and facultatively heterofermentative lactobacilli in pig faeces

Identification and characterization of obligately homofermentative and facultatively heterofermentative strains of Lactobacillus spp. isolated from the faeces of pigs that had been raised under different conditions. The phenotypic relatedness of the isolated strains and reference strains were determined by numerical analysis of total soluble cell protein patterns and simple physiological and biochemical tests. Of the 23 strains isolated from faeces, nine were obligately homofermentative and 14 facultatively heterofermentative. The strains clustered at r > or = 0.61 with Lactobacillus amylovorus (seven strains), Lactobacillus crispatus (one strain), Lactobacillus plantarum (14 strains) and Lactobacillus intestinalis (one strain). Results obtained from the physiological and biochemical tests confirmed the identity of the isolates as determined by numerical analysis of total soluble cell protein profiles. This is the first report on the association of Lact. crispatus and Lact. intestinalis with the gastro-intestinal tract of pigs.

Cloning and expression of L-asparaginase gene in Escherichia coli.

The L-asparaginase (ASN) from Escherichia coli AS1.357 was cloned as a DNA fragment generated using polymerase chain reaction technology and primers derived from conserved regions of published ASN gene sequences. Recombinant plasmid pASN containing ASN gene and expression vector pBV220 was transformed in different E. coli host strains. The activity and expression level of ASN in the engineering strains could reach 228 IU/mL of culture fluid and about 50% of the total soluble cell protein respectively, more than 40-fold the enzyme activity of the wild strain. The recombinant plasmid in E. coli AS1.357 remained stable after 72 h of cultivation and 5 h of heat induction without selective pressure. The ASN gene of E. coli AS1.357 was sequenced and had high homology compared to the reported data.

Lactobacillus fornicalis sp. nov., isolated from the posterior fornix of the human vagina.

Twelve strains isolated from the posterior fornix fluid of the human vagina were identified as Lactobacillus johnsonii, Lactobacillus acidophilus, Lactobacillus gallinarum and Lactobacillus crispatus based on numerical analyses of total soluble cell protein profiles and randomly amplified polymorphic DNA (RAPD)-PCR banding patterns. Five strains grouped with the type strains of Lactobacillus gasseri (DSM 20077T) and Lactobacillus jensenii (DSM 20557T) at r > or = 0.83 in one protein profile cluster, well separated from the other species included in this study. However, numerical analysis of the RAPD-PCR banding patterns of representative strains selected from the L. gasseri-L. jensenii protein cluster clearly indicated that they belong to two different species. Four strains (TV 1010, TG 1013, TV 1018T and TV 1045) grouped into another well separated protein profile cluster at r > or = 0.87. Strains selected from this cluster displayed very similar RAPD-PCR banding patterns and clustered at R2 > or = 0.78, separate from the other strains examined. Sequencing of the 16S rRNA of two representative strains, TV 1018T and TG 1013, of this group indicated that it represents a new member of rRNA group I Lactobacillus, which includes Lactobacillus delbrueckii, the type of the genus, and close relatives Lactobacillus acetotolerans, Lactobacillus kefiranofaciens, Lactobacillus iners, L. jensenii, L. crispatus, L. acidophilus, Lactobacillus helveticus, Lactobacillus amylovorus, Lactobacillus hamsteri, L. johnsonii, L. gasseri and Lactobacillus amylolyticus. The name Lactobacillus fornicalis sp. nov. is proposed for strains TV 1010 (DSM 13172), TG 1013, TV 1018T and TV 1045, with strain TV 1018T (= DSM 13171T = ATCC 700934T) as the type.

Affinity-purification of a TMV-specific recombinant full-size antibody from a transgenic tobacco suspension culture

A TMV-specific full-size murine IgG-2b/κ antibody (mAb24) was expressed in a Nicotiana tabacum cv. Petite Havana SR1 suspension culture (P9s), which was derived from a stably transformed transgenic plant (P9). The integration of an N-terminal murine leader peptide directed the assembled immunoglobulin for secretion. However, in suspension culture, the full-size recombinant antibody, rAb24, was retained by the plant cell wall and was not present in the culture medium. rAb24 expression reached a basal level of 15 μg per gram wet cell weight, corresponding to 0.3% of the total soluble plant cell protein. The level of rAb24 could be increased three-fold by amino acid supplementation of the culture medium. For purification of the recombinant antibody from batch-cultured tobacco suspension cells, the primary plant cell wall was partially digested by enzymatic treatment. This resulted in a total release of recombinant full-size rAb24 into the extraction buffer. A three-step procedure was used to purify the immunoglobulins, starting with cross-flow filtration (step 1) followed by protein A affinity chromatography (step 2) and gel filtration as a final purification step (step 3). This procedure gave a recovery of more than 80% of the expressed rAb24 from plant cell extracts. SDS-PAGE, IEF and immunoblot analyses demonstrated a high degree of homogeneity for the affinity-purified rAb24. An ELISA procedure demonstrated that the specificity and affinity of the protein A affinity purified antibody was indistinguishable from its murine counterpart, indicating the potential of plant cell suspension cultures as bio-reactors for the production of recombinant antibodies.

Lactobacillus species identification, H2O2 production, and antibiotic resistance and correlation with human clinical status.

Lactobacilli recovered from the blood, cerebrospinal fluid, respiratory tract, and gut of 20 hospitalized immunocompromised septic patients were analyzed. Biochemical carbohydrate fermentation and total soluble cell protein profiles were used to identify the species. Hydrogen peroxide production was measured. Susceptibility to 19 antibiotics was tested by a diffusion method, and the MICs of benzylpenicillin, amoxicillin, imipenem, erythromycin, vancomycin, gentamicin, and levofloxacin were determined. A small number of species produced H2O2, and antibiotic susceptibilities were species related. Eighteen (90%) of the isolates were L. rhamnosus, one was L. paracasei subsp. paracasei, and one was L. crispatus. L. rhamnosus, L. paracasei subsp. paracasei isolates, and the type strains were neither H2O2 producers nor vancomycin susceptible (MICs, >/=256 microgram/ml). L. crispatus, as well as most of the type strains of lactobacilli which belong to the L. acidophilus group, was an H2O2 producer and vancomycin susceptible (MICs, <4 microgram/ml).

Isolation and Identification of Sulphite- and Iron Reducing, Hydrogenase Positive Facultative Anaerobes from Cooling Water Systems

Summary The significance of sulphidogenic facultative anaerobes in microbially influenced corrosion (MIC) has been overshadowed by extensive research on sulphate-reducing bacteria (SRB). An enrichment procedure with Modified Iron Sulphite (MIS) medium was employed to select for sulphidogenic facultative anaerobes from industrial cooling water systems. All isolates reduced sulphite and ferric iron, oxidised cathodic hydrogen on mild steel, and some oxidised cathodic hydrogen on stainless steel (3CR12). Facultative anaerobes generating sulphide from sulphite were identified by making use of biochemical tests, API 20 NE, BIOLOG, SDS-PAGE of the total soluble cell proteins, partial sequences of the 16S rRNA gene (rDNA) and RFLP-PCR. Our results show that Aeromonas isolated from cooling water systems, ie A. veronii biotype sobria, A. hydrophila and A. media can reduce both ferric to ferrous iron and sulphite to hydrogen sulphide in IS medium. The results further showed that these sulphidogenic bacteria are all capable of reducing cathodic hydrogen and therefore play a role in MIC.