Relative Amino Acid Composition Research Articles

Effect of Processing Methods on Amino Acid and Fatty Acid Composition of Parkia biglobosa Seeds

Parkia biglobosa (PB) is among the various underexploited nutritious plants. A comparative study was conducted on the effect processing PB has on the amino and fatty acid composition. Fermented (FPB), defatted (DPB), and protein isolates (PI) samples were prepared from Parkia biglobosa seeds using various processing methods, and evaluated using standard analytical protocols. The PI showed the highest significant total non-essential amino acids, essential amino acids, and conditionally essential amino acids when compared with FPB and DPB. In addition, the PI showed the highest significant (p < 0.05) total neutral amino acids, basic amino acids, total aromatic amino acids, and the lowest percentage cystine ratio of total sulphur amino acids and total amino acids in comparison with FPB and DPB. The PI also showed a better-predicted protein efficient ratio, essential amino acid index, biological value, protein content, and nutritional index than FPB and DPB. Furthermore, PI amino acid composition was compared favourably with the reference scores for a whole hen’s egg, preschool child, and provisional scoring pattern. Arginine and histidine values of PI were higher than the recommended values. The FPB (42.29%) showed the highest polyunsaturated fatty acids compared with the DPB (41.94%) and PI (26.7%). The dominant saturated, monounsaturated, and polyunsaturated fatty acids were stearic acid (15.1 DPB), linoleic acid (42.29 DPB), and oleic acid (14.05 PI). The DPB (1.84) showed a better polyunsaturated to saturated (P:S) ratio than FPB (1.74) and PI (0.91). The results revealed that the processing methods improved the relative amino acid composition, whereas the fatty acid composition improved in DPB. Therefore, PI could serve as an alternative in the formulation of complementary foods.

Comparative assessment of amino acids composition in two types of marine fish silage

Fish silage is a brown liquefied product achieved by the action of enzymes when finely grounded whole/parts of either single or mixed fish types are subjected to acidification. This study made a comparative assessment of biochemical and nutritive properties, especially the amino acid composition in supernatant phase of formic acid silages prepared from two fish types, Indian mackerel (Rastrelliger kanagurta) and false travely (Lactarius lactarius) representing fat fish (FF, fat content > 5%) and lean fish (LF, fat content < 5%), respectively during 35 days of fermentation (DoF). Significantly higher content of total amino acid (TAA) and free amino acids (FAA) were recorded in FFS (TAA, 41.2 ± 0.03 mg/g; FAA, 31.3 ± 0.003 mg/g) compared to LFS (TAA, 35.8 ± 0.07 mg/g; FAA, 18.26 ± 0.003 mg/g; FAA, 31.3 ± 0.003 mg/g) (p < 0.05). At the end of 35 DoF, the concentrations of amino acids such as asparagine, histidine, isoleucine, valine, cysteine, serine, lysine and arginine were significantly higher in FFS as compared to LFS. The relative amino acid composition of FFS and LFS varied in accordance with DoF and the relationship was found to be highly significant (ANOVA, p < 0.00001). High concentrations of l-amino acids such as leucine, glutamic acid and arginine were recorded in both FFS and LFS. In conclusion, the analysis suggested that a fermentation period of 25–30 days showed a significant effect on the composition of amino acids in both types of ensilage compared to other fermentation periods (p < 0.05). Considering the role of amino acids in enhancing the plant growth and proliferation, the findings of the present study are quite useful.

Ocean acidification modifies biomolecule composition in organic matter through complex interactions

The main source of marine organic carbon (OC) is autotrophic production, while heterotrophic degradation is its main sink. Increased anthropogenic CO2 release leads to ocean acidification and is expected to alter phytoplankton community composition, primary production rates and bacterial degradation processes in the coming decades with potential consequences for dissolved and particulate OC concentration and composition. Here we investigate effects of increased pCO2 on dissolved and particulate amino acids (AA) and carbohydrates (CHO), in arctic and sub-arctic planktonic communities in two large-scale mesocosm experiments. Dissolved AA concentrations responded to pCO2/pH changes during early bloom phases but did not show many changes after nutrient addition. A clear positive correlation in particulate AA was detected in post-bloom phases. Direct responses in CHO concentrations to changing pCO2/pH were lacking, suggesting that observed changes were rather indirect and dependent on the phytoplankton community composition. The relative composition of AA and CHO did not change as a direct consequence of pCO2 increase. Changes between bloom phases were associated with the prevailing nutrient status. Our results suggest that biomolecule composition will change under future ocean conditions but responses are highly complex, and seem to be dependent on many factors including bloom phase and sampling site.

What CHO is made of: Variations in the biomass composition of Chinese hamster ovary cell lines

BackgroundCell line-specific, genome-scale metabolic models enable rigorous and systematic in silico investigation of cellular metabolism. Such models have recently become available for Chinese hamster ovary (CHO) cells. However, a key ingredient, namely an experimentally validated biomass function that summarizes the cellular composition, was so far missing. Here, we close this gap by providing extensive experimental data on the biomass composition of 13 parental and producer CHO cell lines under various conditions. ResultsWe report total protein, lipid, DNA, RNA and carbohydrate content, cell dry mass, and detailed protein and lipid composition. Furthermore, we present meticulous data on exchange rates between cells and environment and provide detailed experimental protocols on how to determine all of the above. The biomass composition is converted into cell line- and condition-specific biomass functions for use in cell line-specific, genome-scale metabolic models of CHO. Finally, flux balance analysis (FBA) is used to demonstrate consistency between in silico predictions and experimental analysis. ConclusionsOur study reveals a strong variability of the total protein content and cell dry mass across cell lines. However, the relative amino acid composition is independent of the cell line and condition and thus needs not be explicitly measured for each new cell line. In contrast, the lipid composition is strongly influenced by the growth media and thus will have to be determined in each case. These cell line-specific variations in biomass composition have a small impact on growth rate predictions with FBA, as inaccuracies in the predictions are rather dominated by inaccuracies in the exchange rate spectra. Cell-specific biomass variations only become important if the experimental errors in the exchange rate spectra drop below twenty percent.

Purification, characterization and end product analysis of dextran degrading endodextranase from Bacillus licheniformis KIBGE-IB25

Degradation of high molecular weight dextran for obtaining low molecular weight dextran is based on the hydrolysis using chemical and enzymatic methods. Current research study focused on production, purification and characterization of dextranase from a newly isolated strain of Bacillus licheniformis KIBGE-IB25. Dextranase was purified up to 36 folds with specific activity of 1405U/mg and molecular weight of 158kDa. It was found that enzyme performs optimum cleavage of dextran (5000Da, 0.5%) at 35°C in 15min at pH 4.5 with a Km and Vmax of 0.374mg/ml and 182µmol/min, respectively. Relative amino acid composition analysis of purified enzyme suggested the presence of higher number of hydrophobic, acidic and glycosylation promoting amino acids. The N-terminal sequence of dextranase KIBGE-IB25 was AYTVTLYLQG. It exhibited distinct amino acid sequence yet shared some inherent characteristics with glycosyl hydrolases (GH) family 49 and also testified the presence of O-glycosylation at N-terminal end.

Construction of a Large Synthetic Human Fab Antibody Library on Yeast Cell Surface by Optimized Yeast Mating

Yeast surface-displayed antibody libraries provide an efficient and quantitative screening resource for given antigens, but suffer from typically modest library sizes owing to low yeast transformation efficiency. Yeast mating is an attractive method for overcoming the limit of yeast transformation to construct a large, combinatorial antibody library, but the optimal conditions have not been reported. Here, we report a large synthetic human Fab (antigen binding fragment) yeast surface-displayed library generated by stepwise optimization of yeast mating conditions. We first constructed HC (heavy chain) and LC (light chain) libraries, where all of the six CDRs (complementarity-determining regions) of the variable domains were diversified mimicking the human germline antibody repertoires by degenerate codons, onto single frameworks of VH3-23 and Vkappa1-16 germline sequences, in two haploid cells of opposite mating types. Yeast mating conditions were optimized in the order of cell density, media pH, and cell growth phase, yielding a mating efficiency of ~58% between the two haploid cells carrying HC and LC libraries. We constructed two combinatorial Fab libraries with CDR-H3 of 9 or 11 residues in length with colony diversities of more than 10(9) by one round of yeast mating between the two haploid HC and LC libraries, with modest diversity sizes of ~10(7). The synthetic human Fab yeast-displayed libraries exhibited relative amino acid compositions in each position of the six CDRs that were very similar to those of the designed repertoires, suggesting that they are a promising source for human Fab antibody screening.

Damage to Skin Extracellular Matrix Induced by UV Exposure

Chronic exposure to environmental ultraviolet radiation (UVR) plays a key role in both photocarcinogenesis and induction of accelerated skin aging. Although the spatiotemporal consequences of UVR exposure for the composition and architecture of the dermal extracellular matrix (ECM) are well characterized, the pathogenesis of photoaging remains poorly defined. Given the compelling evidence for the role of reactive oxygen species (ROS) as mediators of photoaging, UVR-exposed human skin may be an accessible model system in which to characterize the role of oxidative damage in both internal and external tissues. Although the cell-mediated degradation of dermal components via UVR-induced expression of ECM proteases has long been identified as an integral part of the photoaging pathway, the relative importance and identity of cellular and extracellular photosensitizers (direct hit and bystanders models, respectively) in initiating this enzymatic activity is unclear. Recently, both age-related protein glycation and relative amino-acid composition have been identified as potential risk factors for photo-ionization and/or photo-sensitization. Here, we propose a selective multi-hit model of photoaging. Bioinformatic analyses can be employed to identify candidate UVR targets/photosensitizers, but the action of UVR on protein structure and/or ROS production should be verified experimentally. Crucially, in the case of biochemically active ECM components such as fibronectin and fibrillin, the downstream effects of photo-degradation on tissue homeostasis remain to be confirmed. Both topical antioxidants and inhibitors of detrimental cell signaling may be effective in abrogating the effects of specific UVR-mediated protein degradation in the dermis.

Relative Amino Acid Composition Signatures of Organisms and Environments

BackgroundIdentifying organism-environment interactions at the molecular level is crucial to understanding how organisms adapt to and change the chemical and molecular landscape of their habitats. In this work we investigated whether relative amino acid compositions could be used as a molecular signature of an environment and whether such a signature could also be observed at the level of the cellular amino acid composition of the microorganisms that inhabit that environment.Methodologies/Principal FindingsTo address these questions we collected and analyzed environmental amino acid determinations from the literature, and estimated from complete genomic sequences the global relative amino acid abundances of organisms that are cognate to the different types of environment. Environmental relative amino acid abundances clustered into broad groups (ocean waters, host-associated environments, grass land environments, sandy soils and sediments, and forest soils), indicating the presence of amino acid signatures specific for each environment. These signatures correlate to those found in organisms. Nevertheless, relative amino acid abundance of organisms was more influenced by GC content than habitat or phylogeny.ConclusionsOur results suggest that relative amino acid composition can be used as a signature of an environment. In addition, we observed that the relative amino acid composition of organisms is not highly determined by environment, reinforcing previous studies that find GC content to be the major factor correlating to amino acid composition in living organisms.

Plant growth, metabolism and adaptation in relation to stress conditions. XXVII. Can ascorbic acid modify the adverse effects of NaCl and mannitol on amino acids, nucleic acids and protein patterns in Vicia faba seedlings?

The adverse effects of either NaCl or mannitol on amino acids, protein patterns and nucleic acids in Vicia faba seeds were investigated. The exogenous addition of 4 mM ascorbic acid to the stressing media in which the broad bean seeds were germinated in combination with either the ionic (NaCl) or osmotic (mannitol) stressor induced significant protective changes in the total amount and in the relative composition of amino acids in general and in proline, glycine, glutamic, aspartic, alanine and serine in particular. It also induced changes in nucleic acids (RNA and DNA) content. These changes occurred throughout the entire period of the experiments (12 days). Separate administration of NaCl or mannitol enhanced the occurrence of particular novel proteins that were not detected in control bean seeds (water medium). Protein banding patterns of broad bean seedlings treated with NaCl or mannitol in combination with 4 mM ascorbic acid showed different de novo protein bands, with different molecular weights, at different stages of seedlings growth, with lower levels or a nearly complete absence of the major stress proteins. The pattern of changes for amino acids and nucleic acids and the range of protein bands extracted from the variously treated broad bean seedlings indicate a positive role of ascorbic acid in the alleviation of the damage effects induced by NaCl and mannitol. The importance of this role in the stress tolerance of broad beans is discussed.

Amino acid composition and functional properties of collagen polypeptide from Yak (Bos grunniens) bone

Abstract Collagen polypeptide was prepared from Yak bone by applying papain as a catalyst. Its relative molecular weight distribution and amino acid composition were determined. Moreover, its main functional properties including water and oil absorption, water-holding capacity, emulsifying ability and stability, foaming ability and foam stability were investigated. The polypeptide had a molecular weight distribution of 11.7–43 kDa, in which most distributed at 11.7–25.3 kDa. It was high in glycine, alanine, proline, hydroxyproline and glutamic acid but low in methionine, tyrosine and histidine, the total amino acid amount being 88.06 g/100 g. The polypeptide showed good water adsorption, water-holding capacity, oil absorption, emulsifying and foaming properties. It is hoped to be applied in food and cosmetic.

Modification of fetal plasma amino acid composition by placental amino acid exchangers in vitro

Fetal growth is dependent on both the quantity and relative composition of amino acids delivered to the fetal circulation, and impaired placental amino acid supply is associated with restricted fetal growth. Amino acid exchangers can alter the composition, but not the quantity, of amino acids in the intra- and extracellular amino acid pools. In the placenta, exchangers may be important determinants of the amino acid composition in the fetal circulation. This study investigates the substrate specificity of exchange between the placenta and the feto-placental circulation. Maternal-fetal transfer of radiolabelled amino acids and creatinine were measured in the isolated perfused human placental cotyledon. Transfer of L-[14C]serine or L-[14C]leucine, and [3H]glycine, were measured in the absence of amino acids in the fetal circulation (transfer by non-exchange mechanisms) and following 10-20 micromol boluses of unlabelled amino acids into the fetal circulation to provide substrates for exchange (transfer by exchange and non-exchange mechanisms). The ability of fetal arterial boluses of L-alanine and L-leucine to stimulate release of amino acids from the placenta was also determined using HPLC in order to demonstrate the overall pattern of amino acid release. Experiments with radiolabelled amino acids demonstrated increased maternal-fetal transfer of L-serine and L-leucine, but not glycine, following boluses of specific amino acids into the fetal circulation. L-[14C]Leucine, but not L-[14C]serine or [3H]glycine, was transferred from the maternal to the fetal circulation by non-exchange mechanisms also (P<0.01). HPLC analysis demonstrated that fetal amino acid boluses stimulated increased transport of a range of different amino acids by 4-7 micromol l(-1) (P<0.05). Amino acid exchange provides a mechanism to supply the fetus with amino acids that it requires for fetal growth. This study demonstrates that these transporters have the capacity to exchange micromolar amounts of specific amino acids, and suggests that they play an important role in regulating fetal plasma amino acid composition.

The contributions of specific amino acid side chains to signal intensities of peptides in matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of complex peptide mixtures is often hampered by signal suppression effects as well as certain intrinsic properties of specific peptides that influence the desorption/ionization behavior. The present systematic study reports on the relationship between the occurrence of certain amino acids in peptides and the intensities of the related ions which appear during MALDI-MS analysis for both tryptic digests of proteins and synthetic peptide mixtures. The analysis of the tryptic digests revealed that the peptide sequences of the most intense peaks detected by MALDI-MS contained significantly higher proportions of arginine, phenylalanine, proline, and leucine than the average values for the measured proteins. The relationship between the relative signal intensities and amino acid compositions of peptides was studied in more detail by the partial least squares (PLS) method using equimolar mixtures of 144 well-characterized synthetic peptides. The regression coefficients clearly indicated that the presence of arginine, phenylalanine, leucine and proline tend to enhance the desorption/ionization process which results in higher MALDI-MS peak intensities. Furthermore, it was shown that the impact of arginine depends strongly on the identity of adjacent amino acids.

Markers and signals associated with nitrogen assimilation in higher plants.

A key concept underpinning current understanding of the carbon/nitrogen (C/N) interaction in plants is that the capacity for N assimilation is aligned to nutrient availability and requirements by the integrated perception of signals from hormones, nitrate, sugars, organic acids, and amino acids. Studies on the nature and integration of these signals over the last ten years has revealed a complex network of controls brokered by an interplay of C and N signals. These controls not only act to orchestrate the relative rates of C and N assimilation and carbohydrate and amino acid production, but they also have a significant influence on plant development. Amino acids are the hub around which the processes of N assimilation, associated C metabolism, photorespiration, export of organic N from the leaf, and the synthesis of nitrogenous end-products revolve. Since specific major amino acids or their relative ratios are modulated differentially by photorespiration and N assimilation, even though these processes are tightly intermeshed, they are potentially powerful markers for metabolite profiling and metabolomics approaches to the study of plant biology. Moreover, while minor amino acids show marked diurnal rhythms, their contents fluctuate in a co-ordinated manner. It is probable that factors associated with early events and processes in C and N assimilation influence the relative composition of minor amino acids.

Proximate composition and mineral content of two edible species of Cnidoscolus (tree spinach).

Proximate composition and mineral content of raw and cooked leaves of two edible tree spinach species (Cnidoscolus chayamansa and C. aconitifolius), known locally as 'chaya', were determined and compared with that of a traditional green vegetable, spinach (Spinicia oleraceae). Results of the study indicated that the edible leafy parts of the two chaya species contained significantly (p<0.05) greater amounts of crude protein, crude fiber, Ca, K, Fe, ascorbic acid and beta-carotene than the spinach leaf. However, no significant (p>0.05) differences were found in nutritional composition and mineral content between the chaya species, except minor differences in the relative composition of fatty acids, protein and amino acids. Cooking of chaya leaves slightly reduced nutritional composition of both chaya species. Cooking is essential prior to consumption to inactivate the toxic hydrocyanic glycosides present in chaya leaves. Based on the results of this study, the edible chaya leaves may be good dietary sources of minerals (Ca, K and Fe) and vitamins (ascorbic acid and beta-carotene).

Phosphate availability in combination with nitrate availability affects root yield and chicon yield and quality of Belgian endive (Cichorium intybus)

This study investigated the effects of nitrate and phosphate nutrition on chicory tap root development and chicon quality. Plants of chicory (Cichorium intybus flash) were grown on four concentrations of nitrate and phosphate: 3 mM NO3 / 1 mM PO 4 3− , high N and high P (control plants, N / P); 3 mM NO 3 − / 0.05 mM H2PO3−4, high N and low P (N / p); 0.6 mM NO3 / 1 mM PO 4 3− , low N and high P (n / P); 0.6 mM NO 3 − / 0.05 mM PO 4 3− , low N and low P (n / p). The results suggested that, nitrogen limitation had the greatest impact on the shoot/root dry weight ratio. Only small changes in the shoot/root dry weight could be attributed to P limitation alone. Compared with the control, N limitation caused a marked increase in root SST activity (sucrose sucrose fructosyl transferase, the enzyme responsible for fructan synthesis in roots), the effect of P limitation on SST activity was less pronounced. The activity of SS (sucrose synthase) was also noticeably elevated at the early sample data by N limitation. N and P uptake were estimated by the amount of N and P accumulated by the whole plant during the vegetative period. With N limitation, P accumulation was decreased by 40-60% over the experimental period. The effects of P limitation on N accumulation were more variable, N uptake was 60% lower than the control during the tuberizing period (107 days after sowing). With N limitation, P concentrations in roots were lowered by 20-25%. With P limitation, total N concentration in roots decreased by 50% relative to the control, while nitrate concentration was increased more than 8 fold. These effects were detected only at 107 DAS. The amino acid content of roots was not affected by P limitation, however, N limitation altered strongly total amino acids. P limitation did alter the relative amino acid composition of roots early in the vegetative period: Roots harvested at the end of vegetative period were forced in the dark to produce an etiolated bud, the edible chicon. High N and high P fertility (N/P) were associated to a poor chicon yield and quality. However the presence of low P during vegetative growth moderates adverse effects of high nitrate and greatly improved chicon yeild and quality.

Amino acid composition in xylem sap of soybean related to the evaluation of N2fixation by the relative ureide method

Abstract For the estimation of N2 fixation of soybean (Glycine max (L.) Merr.) by the relative ureide method, we modified the original equation [ureide-N/(ureide-N + α-amino-N + nitrate-N)] proposed by Herridge et al. to [ureide-N/(ureide-N + 2 × α-amino-N + nitrate-N)], based on the observation that asparagine which has two N atoms in one molecule was the major amino acid in the xylem sap of soybean plants. However, since the amino acid composition may change with the growth stages or environmental factors such as N availability, it is necessary to determine whether the factor “2×” is appropriate. In the present paper, the experiments were carried out to analyze the levels and relative composition of amino acids and amides in the xylem sap of soybean cv. Enrei which was grown in the field with three fertilizer treatments, i.e. control with a small amount of basal dressing of ammonium sulfate, deep placement or top dressing of coated urea (slow release N fertilizer). Although the composition of the amino ...

Resistance of different host plants and cultivars of cassava to the mealybug Phenacoccus manihoti: Effect of infestation on leaf extract composition

AbstractThe effect of a 2-month infestation by the cassava mealybug (Phenacoccus manihoti Mat. Ferr.) on the metabolism of nitrogen (amino acids) and carbon (carbohydrates), leaf area and total dry weight of five cassava varieties (Manihot esculenta Crantz), faux-caoutchouc (a hybrid of M. esculenta and M. glaziovii Muell, Arg.), poinsettia (Euphorbia pulcherrima Wild) and talinum (Talinum triangulare Jack) was studied. Free amino acid and free sugar contents as well as relative free amino acid composition in the leaf extracts, although found to be very different from one plant to another, were not significantly modified by P. manihoti infestation, except for the total amino acid contents of the cassava variety 30M7. Variations in one particular amino acid induced by mealybug infestation were not linked to the antibiotic resistance of these plants. Infestation by the cassava mealybug did not modify the total dry weight but reduced the total leaf area although this reduction was only significant in cassava varieties 59M2, 30M7 and M'pembe, and in faux-caoutchouc. Within the genus Manihot, the reduction in leaf area was strongly correlated (r= -0.878, P≤0.05) to the degree of antibiotic resistance and was coupled to an increase in the ratio of sugars to amino acids, suggesting a similarity between the effects of water stress and those of mealybug infestation.

Characteristics of six isoperoxidases from Korean radish root

Two cationic isoperoxidases (designated C 1 and C 3) and four anionic isoperoxidases (designated A 1, A 2, A 3n and A 3) from Korean radish ( Raphanus sativus L.) root have been purified to apparent homogeneity, and some of their enzymatic properties were characterized. All six isoperoxidases are glycoproteins composed of a single polypeptide chain. The M r s Of C 1, C 3, A 1 and A 2 were ca 44 000, while anionic isoperoxidase A 3n and A 3 have M r s of 31 000 and 50 000, respectively. Deglycosylated A 2 and C 3 by trifluoromethanesulphonic acid treatment showed M r s of 37 000 and 40 000, respectively, suggesting that the carbohydrate contents for these isoenzymes are 14 and 9%, respectively. Relative amino acid compositions of four anionic isoperoxidases (designated A 1, A 2, A 3n and A 3) and one cationic isoperoxidase C 3 were determined. N-Terminal amino acid sequences were determined for A 1, A 3n and C 3, while A 2 was found to have a blocked amino terminal residue. Kinetic studies with respect to various synthetic and naturally occurring substrates were also investigated.

Chemical inactivation of the Kveim reagent.

In an attempt to identify the nature of the active principle, Kveim reagent was exposed to chemical fractionating agents. Thirty-one patients with sarcoidosis underwent simultaneous intradermal injection with fractionated and unfractionated Kveim material. Kveim reagent was stable in the presence of DNAse, RNAse, pronase, 95% phenol, neutral detergent, and to lipid extraction with chloroform-methanol. Kveim reagent was also stable in the presence of both 8 M urea (8MU) and 2-mercaptoethanol (2ME) when used alone. When both these agents were used together, Kveim reagent was inactivated. Fourteen patients had a positive test to unfractionated Kveim reagent; of these, only 2 gave a positive response to material fractionated by exposure to 8MU and 2ME. Simultaneous exposure to 8MU and 2ME was more likely to inactivate Kveim reagent (10/10 tests) than sequential exposure to 8MU and 2ME (2/4 tests). Chemical analysis of the fractionated material showed that it retained granuloma-generating activity despite the lack of carbohydrates. Protein loss in terms of total and relative amino acid composition was progressive and non-specific throughout processing. These results are consistent with a protein-active principle which is dependent on three-dimensional structure.

Identification, purification, and characterization of major antigenic proteins of Campylobacter jejuni.

Evidence from developing countries and volunteer studies indicates that immunity to Campylobacter jejuni and Campylobacter coli may be acquired, but the antigenic basis for this protection is poorly defined. We have purified to homogeneity four proteins with molecular weights of 28,000 (PEB1), 29,000 (PEB2), 30,000 (PEB3), and 31,000 (PEB4) from epidemic C. jejuni strain 81-176 using acid extraction and sequential ion-exchange, hydrophobic interaction, and gel filtration chromatography. The relative amino acid compositions of these four proteins are similar. NH2-terminal sequence analysis indicates that all four proteins are different, although the first 35 amino acids of PEB2 and PEB3 are 51.4% homologous. Isoelectric focusing showed that all four are basic proteins with pI of 8.5 for PEB1 protein and greater than 9.3 for the others. Use of the purified proteins as antigens in an IgG enzyme-linked immunosorbent assay (ELISA) found that seroconversion to the PEB1 or PEB3 proteins occurred in 15 of 19 patients with sporadic C. jejuni or C. coli infection. In comparison, only two, six, and 14 of these patients seroconverted to PEB2, PEB4, or the acid extract antigen. In an ELISA with whole bacterial cells as antigens, antiserum to the acid-extracted antigens showed broad recognition of C. jejuni, C. coli, C. fetus, C. lari, and Helicobacter pylori. Antiserum to PEB1 recognized all 35 C. jejuni and all 15 C. coli strains but none of the isolates of the other three bacterial species. The PEB1 and PEB3 proteins appear to be candidate antigens for both a Campylobacter vaccine and for serological assays for the pathogen.