Dipartimento di Scienza degli Alimenti, Università degli Studi di Napoli Federico II, Portici, Italy. lilla@unina.it
Mass spectrometry has been used to map chymosin from a fermentative source. The copresence of the two known genetic variants A (Asp(244)) and B (Gly(244)) was ascertained in bovine chymosin. By contrast, either the A or the B genetic variant occurred in the three commercial samples of recombinant calf chymosin (RCC). Specific biomarker proteins were searched to identify the enzyme source, in both bovine chymosin and RCC samples. Analyzing the derived tryptic peptides, evidence was provided that RCC and bovine chymosin are mainly formed by (1-323),(3-323), and (40p-323)(suffix "p" denotes residues in the pro-segment region of chymosin), whereas the minor components,(4-323),(5-323), and (6-323), were only detected in bovine chymosin. Additionally, the three commercial RCC samples contained the protein species (1-323),(38p-323),(39p-323), and (40p-323) and the shorter form (3-323). Differentiation of the natural and bioengineered enzyme is based upon the detection of these unique minor components by mass spectrometry.
Other papers by authors:J Dairy Res. 2004 Feb ;71 (1):14-9 15068061 Cit:2
Lina Chianese, Simonetta Caira, Sergio Lilla, Fabiana Pizzolongo, Pasquale Ferranti, Giovanni Pugliano, Francesco Addeo
Dipartimento di Scienza degli Alimenti, Università degli Studi di Napoli Federico II, Portici, Italy. chianese@unina.it
A novel electrophoretic alpha-lactalbumin (alpha-la) variant was detected in the Italian water buffalo breed. The isoelectric point of the variant, labelled A, was lower than the most frequent variant B. It presented an allelic frequency of 0.5% compared with the 97.1% of the BB allele. From Liquid Chromatography-Electrospray Ionization/Mass spectrometry, the molecular mass of the two alpha-la A and B variants were measured as 14,235.1+/-0.8 and 14,236.1+/-0.9 Da, respectively. The two proteins were sequenced and differentiated from one another by a single amino acid substitution, Asn45(B)-->Asp45(A). As this amino acid substitution altered the N-glycosylation sequence consensus Asn45-X-Ser46 it may be deduced that the protein glycosylation level of the alpha-la A would decrease.
Simonetta Caira, Pasquale Ferranti, Monica Gatti, Maria Emanuela Fornasari, Francesca Barone, Sergio Lilla, Germano Mucchetti, Gianluca Picariello, Lina Chianese, Erasmo Neviani, Francesco Addeo
Istituto di Scienze dell'Alimentazione del C.N.R., 83100 Avellino, Italy. scaira@isa.av.cnr.it
Four Lactobacillus helveticus strains were studied for proteolytic capacity and general aminopeptidase (AP) and X-Pro dipeptidyl aminopeptidase (DAP) activity. The rate of hydrolysis and the activity against synthetic substrates with N-terminal residues of Arg, Lys, Leu, Glu or Pro, varied markedly among the strains. The X-Pro DAP activity was consistently high. The crude cell-wall and cytoplasm extracts from strain Lb. helveticus ISLC59 were analysed thoroughly for their proteolysis ability by using four synthetic peptide substrates, including alpha(s)1-CN(f1-23). Peptides formed during in vitro hydrolysis of the synthetic substrates by cell wall and cytoplasm preparations were identified by LC-ESI/MS. In doing so, it was possible to infer a prevalent endopeptidase activity splitting Lys7-His8 and Gln13-Glu14 bonds in the cytoplasm, and to deduce a secondary activity, which hydrolysed Glu14-Val15, Leu16-Asn17, Glu18-Asn19 and Lys3-His4 bonds lacking in the cell-wall. The presence of exopeptidases, as mainly AP, DAP, and carboxypeptidase (CPase) was deduced from the formation of several N- and C-terminally truncated peptides sets. The AP activity was higher in the cell-wall layer, where CPase activity was absent. The in vitro assays with cell extracts of the Lb. helveticus ISLC59 strain revealed extensive exopeptidase and endopeptidase activities. In several cases, the hydrolytic system of Lb. helveticus that splits in vitro alpha(s)1-CN(f1-23) peptide bonds was similar to that of Lactococcus lactis. The effects were also compared with those occurring in vivo in hard cheese such as Grana Padano.
J Chromatogr A. 2011 Jun 17;: 21737089
Istituto di Scienze dell'Alimentazione (ISA)- CNR, Via Roma 52 A/C, 83100 Avellino, Italy.
In the last years proteomic science has started to provide an important contribution to the disclosure of basic aspects of food-related diseases. Among these, the identification of proteins involved in food allergy and their mechanism of activation of toxicity. Elucidation of these key issues requires the integration of clinical, immunological, genomic and proteomic approaches. These combined research efforts are aimed to obtain structural and functional information to assist the development of novel, more reliable and powerful diagnostic protocols alternative to the currently available procedures, mainly based on food challenge tests. Another crucial aspect related to food allergy is the need for methods to detect trace amounts of allergenic proteins in foods. Mass spectrometry is the only non-immunological method for high-specificity and high-sensitivity detection of allergens in foods. Nowadays, once provided the appropriate sample handling and the correct operative conditions, qualitative and quantitative determination of allergens in foods and ingredients can be efficiently obtained by MALDI-TOF-MS and LC-MS/MS methods, with limits of detection and quantification in the low-ppb range. The availability of accurate and fast alternatives to immunological ELISA tests may also enable the development of novel therapeutic strategies and food processing technologies to aid patients with food allergy or intolerance, and to support allergen labelling and certification processes, all issues where the role of proteomic science is emerging.
Dipartimento di Scienza degli Alimenti-Università di Napoli "Federico II", Parco Gussone, 80055 Portici, Napoli, Italy.
The in-depth characterization of water buffalo (WB) whey proteins based on chromatographic and mass spectrometric techniques revealed unexpected structural co- and post-translational modifications for β-lactoglobulin (β-Lg). The residues Lys⁴⁷ and Lys⁶⁹ of β-Lg were found to be lactosylated early, at the time of milking. Thiol groups of β-Lg underwent a dynamic sulfhydryl/disulfide exchange that is probably essential in accomplishing specific physiological requirements in which proteins may alternatively act either as a trigger or as a target. In this sense, the free sulfhydryl group of β-Lg established a glutathionylation/deglutathionylation equilibrium, which could be functional in conveying and delivering glutathione. Furthermore, the N-lauroylated β-Lg occurring exclusively in WB milk has been characterized for the first time. N-acylation could be an evolutionary remnant of ancestral lipocalins. Combined with the known aptitude of β-Lg to interact with phospholipid bilayers, this suggests that the protein could also be involved in the membrane translocation of small molecules, in addition to targeting, trafficking or the maintenance of membrane integrity. This structural characterization of β-Lg adds to the currently existing data and expands our understanding of the possible biological roles of this enigmatic protein.
Istituto di Scienze dell'Alimentazione, CNR, 83100 Avellino, Italy.
Owing to its extensive use in the human diet, wheat is among the most common causes of food-related allergies and intolerances. Allergies to wheat are provoked by ingestion, inhalation or contact with either the soluble or the insoluble gluten proteins in wheat. Gluten proteins, and particularly the gliadin fraction, are also the main factor triggering celiac disease, a common enteropathy induced by ingestion of wheat gluten proteins and related prolamins from oat, rye and barley in genetically susceptible individuals. The role of gliadin and of its derived peptides in eliciting the adverse reactions in celiac disease are still far from being completely explained. Owing to its unique pathogenesis, celiac disease is widely investigated as a model immunogenetic disorder. The structural characterization of the injuring agents, the gluten proteins, assumes a particular significance in order to deepen the understanding of the events that trigger this and similar diseases at the molecular level. Recent developments in proteomics have provided an important contribution to the understanding of several basic aspects of wheat protein-related diseases. These include: the identification of gluten fractions and derived peptides involved in wheat allergy and intolerance, including celiac disease, and the elucidation of their mechanism of toxicity; the development and validation of sensitive and specific methods for detecting trace amounts of gluten proteins in gluten-free foods for intolerant patients; and the formulation of completely new substitute foods and ingredients to replace the gluten-based ones. In this article, the main aspects of current and prospective applications of mass spectrometry and proteomic technologies to the structural characterization of gluten proteins and derived peptides are critically presented, with a focus on issues related to their detection, identification and quantification, which are relevant to the biochemical, immunological and toxicological aspects of wheat intolerance.
Carmela De Simone, Pasquale Ferranti, Gianluca Picariello, Ilaria Scognamiglio, Alessandra Dicitore, Francesco Addeo, Lina Chianese, Paola Stiuso
Department of Food Science, University of Naples Federico II, Portici, Naples, Italy.
HASH(0x51bb0b0)
Lina Chianese, Maria Grazia Calabrese, Pasquale Ferranti, Rosalba Mauriello, Giuseppina Garro, Carmela De Simone, Maria Quarto, Francesco Addeo, Gianfranco Cosenza, Luigi Ramunno
Dipartimento di Scienza degli Alimenti, Università degli Studi di Napoli Federico II, Facoltà di Agraria, Via Università 100, Parco Gussone, I-80055 Portici (Napoli), Italy. chianese@unina.it
At present, compared with bovine milk, the characterization of donkey milk caseins is at a relatively early stage progress, and only limited data are related to its genetic polymorphism. In this work, the heterogeneity of donkey caseome was investigated using a proteomic approach, based on one-(PAGE, UTLIEF) and two-dimensional (PAGE-->UTLIEF) electrophoresis, stained with either Coomassie Brilliant Blue or specific polyclonal antibodies, and structural MS analysis. These combined methodologies allowed the contemporary identification of donkey alpha(s1), alpha(s2), beta and kappa-CN with their related heterogeneity due to phosphorylation (alpha(s1), alpha(s2) and beta-CN), glycosylation (kappa-CN) and incorrect splicing of RNA in mRNA (deleted forms of alpha(s1)-CN and beta-CN). The results achieved showed 11 components for kappa-CN, six phosphorylated components for beta and alpha(s1)-CN and three main phosphorylated components for alpha(s2)-CN, each accounting for 10, 11 and 12 P/mole. At this regard, for the first time, the primary structure of the expressed protein corresponding to the only available donkey alpha(s2)-CN cDNA sequence was determined. Furthermore beta-CN was found in homozygous and heterozygous state for the occurrence of a genetic beta-CN variant having a MW value 28 mass units higher than the common beta-CN phenotype.
Istituto di Scienze dell'Alimentazione, Consiglio Nazionale delle Ricerche (CNR), Via Roma 64, 83100 Avellino, Italy. picariello@isa.cnr.it
The potential of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) for the analysis of intact triacylglycerols (TAGs) is generally limited by the extensive in-source prompt fragmentation. The sequential deposition of matrix and TAGs over the stainless steel target precoated with a thin layer of nitrocellulose (NC) drastically reduced fragmentation in the MALDI-TOF MS profiling of oils and fats. The NC MALDI-TOF MS profiles of native and thermally stressed virgin olive oil and butter are reported as case studies, along with test analyses of a standard mixture of mono-, di-, and triacylglycerols. Mass spectra were almost completely devoid of both fragment and matrix ion signals, thus disclosing relevant information, especially in the low molecular mass range. The detection of several partial acylglycerols of low abundance and minor TAGs that are barely observed with other techniques also provided evidence for an increased dynamic range of NC MALDI-TOF MS that was due to the minimization of suppressive effects. The NC film substrate also improved the shot-to-shot and sample-to-sample reproducibility of the ion production through the exhibition of a more homogeneous matrix/analyte cocrystallization, thus enabling MALDI-based measurements to a consistent quantification of TAGs.
Dipartimento di Scienza degli Alimenti - Università di Napoli 'Federico II', Parco Gussone, 80055 Portici (Napoli), Italy.
The possibility of detecting extraneous milk in singles species cheese-milk has been explored. A mass spectrometry (MS)-based procedure has been developed to detect 'signature peptides', corresponding to the predefined subset of 'proteotypic peptides', as matchless analytical surrogates of the parent caseins. Tryptic digests of skimmed milk samples from four species were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Amongst the candidate signature peptides that are able to differentiate milks from the four species, the alpha(s1)-casein (CN) f8-22 peptide was selected as a convenient marker for bovine, ovine and water buffalo milk while the f4-22 peptide was selected as a marker for the two caprine alpha(s1)-CN A and B variants, which differ by a Pro(16)(B)->Leu(16)(A) substitution. MALDI analysis of the digest allowed the detection of alpha(s1)-CN f8-22 and caprine alpha(s1)-CN f4-22. The accurate evaluation of caprine milk in a quaternary mixture required the development of a liquid chromatography/electrospray ionization (LC/ESI)-MS procedure. Five synthetic signature peptide analogues, which differed from their natural counterparts by a single amino acid substitution, were used as internal standards to quantify the alpha(s1)-CN, which was chosen as a reference milk protein, from the different species. The limits of detection were 0.5%(1% for caprine) for either the MALDI or the LC/ESI-MS method. The isotopic-label-free quantification of isoform- or variant-specific signature peptides has disclosed a convenient approach for targeting proteins in complex mixtures.
Gianluca Picariello, Pasquale Ferranti, Olga Fierro, Gianfranco Mamone, Simonetta Caira, Aldo Di Luccia, Stefano Monica, Francesco Addeo
Istituto di Scienze dell'Alimentazione-CNR, Via Roma 52 A/C, I-83100 Avellino, Italy. picariello@isa.cnr.it
Resistance to proteases throughout the gastrointestinal (GI) tract is a prerequisite for milk-derived peptides to exert biological activities. In this work an in vitro multi-step static model to simulate complete digestion of the bovine milk proteins has been developed. The experimental set-up involved the sequential use of:(i) pepsin,(ii) pancreatic proteases, and (iii) extracts of human intestinal brush border membranes, in simulated gastric, duodenal and jejuneal environments, respectively. Enzymatic concentrations and reaction times were selected in order to closely reproduce the in vivo conditions. The aim was to identify the peptide candidates able to exhibit significant bioactive effects. Casein and whey protein peptides which survived the in vitro GI digestion have been identified by the combined application of HPLC and mass spectrometry techniques. While the permanence of the main potentially bioactive peptides from both casein and whey proteins was found of limited physiological relevance, the high resistance to proteolysis of specific regions of beta-lactoglobulin (beta-Lg), and especially that of the peptide beta-Lg f125-135, could have implications for the immunogenic action of beta-Lg in the insurgence of cow's milk allergy.
Latest similar papers: Jaewon Kim, Laurie Jones, Lisa Taylor, Gunasekaran Kannan, Frank Jackson, Hollis Lau, Ramil F Latypov, Bob Bailey
Department of Analytical & Formulation Sciences, 1201 Amgen Court West, Seattle, WA 98119-3105, USA. jaewonk@amgen.com
The unique cation exchange chromatography (CEX) charge variant profile of mAb1 is characterized by a combination of mass spectrometry, limited Lys-C digestion followed by CEX separation and structural analysis. During CEX method development, mAb1 showed several unexpected phenomena, including a unique profile containing two main species (acidic 2 and main) and significant instability during stability studies of the main species. Reduced Lys-C peptide mapping identified a small difference in one of the heavy chain peptides (H4) in acidic 2 and further mass analysis identified this difference as Asn55 deamidation. However, the amount of Asn55 deamidation in acidic 2 could account for only half of the species present in this peak. Lys-C limited digest followed by CEX separated several unique peaks in the acidic peak 2 including two pre Fab peaks (LCC1 and LCC2). Whole protein mass analysis suggested that both LCC1 and LCC2 were potentially deamidated species. Subsequent peptide mapping with MS/MS determined that LCC1 contained isoAsp55 and LCC2 contained Asp55. Combining LCC1 and LCC2 CEX peak areas could account for nearly all of the species present in acidic peak 2. Subsequent detailed sequence analysis combined with molecular modeling identified Asn55 and its surrounding residues are responsible for the different CEX behavior and instability of mAb1 following forced degradation at high pH. Overall, the combinatorial approach used in this study proved to be a powerful tool to understand the unique charge variant and stability profile of a monoclonal antibody.
Molecular Biology Unit, Division of Dairy Microbiology, National Dairy Research Institute, Karnal, Haryana, India. ashwanindri@rediffmail.com
Calf rennet, which consists of over 90% chymosin, is commonly used in cheese industries for the curdling of milk. Various animal, plant and microbial sources have been exploited as possible alternatives to calf rennet. The coagulating properties of the enzymatic preparations (coagulants) from these sources differ in terms of their physicochemical factors. The cheese industry has always sought out novel and stable enzyme sources, and recombinant chymosin has been found to be an effective alternative since it possesses several advantages over plant and microbial milk-clotting enzymes. This paper reviews the use of various milk coagulants, especially animal coagulants, for cheese making. Advancements in genetic and protein engineering to produce recombinant chymosin are discussed in addition to evaluating its identity to the rennet available from natural sources.
Mirna Brkljacić, Vesna Matijatko, Ivana Kis, Nada Kucer, Jadranka Forsek, Renata Barić Rafaj, Darko Grden, Marin Torti, Iva Mayer, Vladimir Mrljak
University of Zagreb Clinic for Internal Diseases Heinzelova 55 10 000 Zagreb Croatia.
The aim of the present study was to detect and characterise the species and subspecies of Babesia spp. that cause canine babesiosis in Croatia. Twenty-eight dogs with typical signs of babesiosis (lethargy, anorexia, fever, dark urine and thrombocytopenia) were included in this study. Their blood smears showed the presence of Babesia canis . The results showed the detection of one subspecies, namely Babesia canis canis using PCR, and subsequent sequence analysis demonstrated portions of the nss rRNA gene in 27 out of 28 samples. Sequence analysis of the isolates showed 100% identity in 11 samples, 99.7% identity (one nucleotide difference) in 11 samples and 99.4% identity (two nucleotides difference) in 5 samples with B. canis canis . The results of this study confirm the presence of B. canis canis in infected dogs in Croatia and demonstrate a slightly new genetic variant of Babesia subspecies.
Centro de Investigación en Alimentación y Desarrollo, AC, Hermosillo, Sonora, Mexico. vallejo@cascabel.ciad.mx
This report presents an analytical strategy for the identification of beta-lactoglobulin (beta-Lg) variants or isoforms in bovine milk by using capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) and nano-liquid chromatography-electrospray-mass spectrometry-mass spectrometry (nano-LC-ESI-MS-MS). beta-Lg isoforms were separated and collected by CE, and their molecular masses were determined by CE-ESI-MS. Protein isoforms collected by CE were digested with trypsin and analyzed by nano-LC-ESI-MS-MS. Isoforms analyzed by CE-ESI-MS presented multiply charged ions that after deconvolution resulted in molecular masses of 18,364 +/- 1.0 Da and 18,279 +/- 2.0 Da. Fragmentation of parent ions in MS-MS experiments of specific tryptic peptides were carried out to identify beta-Lg variants in milk samples. The milk samples analyzed were indeed phenotypes AA, BB, or AB.
Dipartimento di Scienze Chimiche, Università degli Studi di Catania, Viale A. Doria 6, I‐95125 Catania, Italy.
The sequence determination of a new variant of beta-LG II, detected as a minor component by reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS) analysis of the whey fraction from a milk sample taken from an individual donkey belonging to the 'Ragusana' species of eastern Sicily, is reported. Direct RP-HPLC/ESI-MS analysis of the whey fraction from this milk sample allowed the identification of a new variant of beta-LG II, based on the determination of the M(r) of the intact protein. The new protein, with an experimentally determined M(r) of 18311 Da, was detected as a minor component in the whey fraction investigated. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)MS and RP-HPLC/ESI-MS/MS analyses of the tryptic digest of the new protein demonstrate that it presents two amino acid substitutions with respect to the sequence of beta-LG II A, namely a substitution Pro-->Cys at position 110, and a substitution Asp-->Gly at position 162. The disulfide bonds between the four cysteines, not directly determined in donkey's and horse's beta-LG II, were shown to occur between Cys(106)-Cys(120) and Cys(66)-Cys(161), as in other mammalian beta-LGs. The new beta-LG II variant from donkey was named D. Copyright (c) 2007 John Wiley & Sons, Ltd.
Mass Spectrometry Resource, Department of Biochemistry, Boston University School of Medicine, 715 Albany Street R806, Boston, MA 02118, USA.
A nonenzymatic posttranslational modification of proteins and peptides is the spontaneous deamidation of asparaginyl residues via a succinimide intermediate to form a varying mixture of aspartyl and isoaspartyl residues. The isoaspartyl residue is generally difficult to detect particularly using mass spectrometry because isoaspartic acid is isomeric with aspartic acid so that there is no mass difference. However, electron capture dissociation has demonstrated the ability to differentiate the two isoforms in synthetic peptides using unique diagnostic ions for each form; the c(r)(*)+ 58 and z(l-r)- 57 fragment ions for the isoAsp form and the Asp side chain loss ((M + nH)(n-1)(+*)- 60) for the Asp form. Shown here are three examples of isoaspartyl detection in peptides from proteins; a deamidated tryptic peptide of cytochrome c, a tryptic peptide from unfolded and deamidated ribonuclease A, and a tryptic peptide from calmodulin deamidated in its native state. In all cases, the c(r)(*)+ 58 and z(l-r)- 57 ions allowed the detection and localization of isoaspartyl residues to positions previously occupied by asparaginyl residues. The (M + nH)(n-1)(+*)- 60 ions were also detected, indicating the presence of aspartyl residues. Observation of these diagnostic ions in peptides from proteins shows that the method is applicable to defining the isomerization state of deamidated proteins.
Hum Genet. 2005 Aug ;117 (4):349-56 15915326 Cit:8
Lambertus Klei, Silviu-Alin Bacanu, Marina Myles-Worsley, Brandi Galke, Weiting Xie, Josepha Tiobech, Caleb Otto, Kathyrn Roeder, Bernie Devlin, William Byerley
Department of Statistics, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
We report on linkage analysis of a completely ascertained population of familial psychosis derived from the oceanic nation of Palau. Palau, an archipelago of islands in the Southern Pacific, currently has a population of approximately 23,000 individuals. The peoples of Palau populated these islands recently in human history, approximately 2,000 years ago. As both historical and genetic evidence suggest, the population is far more homogeneous than most other populations undergoing genetic studies, and should therefore prove quite useful for mapping genetic variants having a meaningful impact on susceptibility to psychotic disorders. Moreover, for our study, essentially all on-island schizophrenics (150) and individuals with other psychotic disorders (25) participated. By analysis of narrow (only schizophrenia) and broad (all psychosis) diagnostic schemes, two-point linkage analyses suggest that two regions of the genome harbor genetic variants affecting liability in most families, 3q28 (LOD = 3.03) and 17q32.2 (LOD = 2.80). Results from individual pedigrees also support 2q37.2, 2p14, and 17p13 as potentially harboring important genetic variants. Most of these regions have been implicated in other genetic studies of psychosis in populations physically quite distant from this Oceanic population, although some (e.g., 3q28) appear to be novel results for schizophrenia linkage analyses.
Department of Parasitology, Leiden University Medical Centre, Leiden, The Netherlands. jj.verweij@lumc.nl
Classically, detection of Entamoeba histolytica is performed by microscopic examination for characteristic cysts and/or trophozoites in fecal preparations. Differentiation of E. histolytica cysts and those of nonpathogenic amoebic species is made on the basis of the appearance and the size of the cysts. However, by classical means objective tools for confirmation and quality control do not exist. Therefore, a reverse line blot hybridization assay was developed to detect a variety of Entamoeba species and genetic variants known to infect humans. The assay was performed after amplification with general Entamoeba-specific primers. The assay could identify four genetic variants of Entamoeba polecki-like cysts as well as E. histolytica, Entamoeba dispar, Entamoeba hartmanni, Entamoeba moshkovskii and Entamoeba coli and even mixed infections in a range of controls and fecal samples. This technique can be used as an additional standard for diagnosis, epidemiology, and quality control for amoebic infections.
Molecular Biology Unit, Dairy Microbiology Division, National Dairy Research Institute, Karnal, India
Bovine chymosin, an aspartyl protease extracted from abomasum of suckling calves, is synthesized in vivo as preprochymosin and secreted as prochymosin which is autocatalytically activated to chymosin. Chymosin is bilobular, with Asp 32 and Asp 215 acting as the catalytic residues. Chymosin A and chymosin B have pH optima of 4.2 and 3.8, respectively, and act to initiate milk clotting by cleaving kappa-casein between Phe 105 and Met 106. The gene encoding chymosin has been cloned and expressed in suitable bacteria and yeast hosts under the control of lac, trp, trp-beta, gly A genes, and serine hydroxymethyl-transferase promoters. Protein engineering of chymosin has also been attempted. A number of companies are now producing recombinant chymosin for commercial use in cheese manufacture.
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-3-1 Maidashi, Higashi-ku, Fukuoka 812-8582.
A method was established for evaluating Asn deamidation and Asp isomerization/racemization. To detect the subtle changes in mass that accompany these chemical modifications, we used a combination of enzyme digestion by endoproteinase Asp-N, which selectively cleaves the N-terminus of L-alpha-Asp, and MALDI/TOF-mass spectrometry. To achieve better resolution, we employed digests of (15)N-labeled protein as an internal standard. To demonstrate the advantages of this method, we applied it to identify deamidated sites in mutant lysozymes in which the Asn residue is mutated to Asp. We also identified the deamidation or isomerization site of the lysozyme samples after incubating them under acidic or basic conditions.
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