Copyright (c) 2009 All rights reserved. http://works.bepress.com/rjimenez Recent documents in Rafael Jiménez-Flores en-us Sun, 31 May 2009 10:56:16 PDT 3600 http://works.bepress.com/rjimenez/48 http://works.bepress.com/rjimenez/48 Fri, 23 May 2008 15:56:13 PDT Effects of heat treatment on skim milk proteins were characterized using an immunoblot method. Proteins in heat-treated skim milk were separated by polyacrylamide gel electrophoresis (nonreducing and reducing conditions) and electrophoretically transferred to nitrocellulose and β-lactoglobulin, α-lactalbumin, and κ-casein were detected by immunoblotting. High molecular weight immunoreactive β-lactoglobulin was apparent in skim milk samples heated at 65°C and above when electrophoresed under nonreducing conditions. Formation of high molecular weight immunoreactive β-lactoglobulin was observed in heat-treated samples electrophoresed under reducing conditions. Immunoreactive α-lactalbumin in skim milk was not substantially affected by heat treatment. High molecular weigh immunoreactive κ-caesin was present in nonreduced samples from each temperature treatment, including incubation at room temperature, but not in samples electrophoresed under reducing conditions. This immunoblot method should be valuable for further study of intermolecular interactions of milk proteins. Walter L. Hurley Dairy Science http://works.bepress.com/rjimenez/44 http://works.bepress.com/rjimenez/44 Fri, 23 May 2008 15:55:41 PDT The bovine β-casein gene, A2 genetic variant, has been mutated at positions 70 and 71 for the introduction of a glycosylation signal (Asn-X-Ser). These mutants have been constructed to study the functionality of β-casein glycosylated exclusively at Asn68. The mutation was generated using PCR-based site-directed mutagenesis, and it was derived from bovine β-casein cDNA. The mutant cDNAs including the wild-type β-casein gene have been subcloned into the yeast Pichia pastoris expression vector pHIL-D2, which contains the methanol-inducible alcohol oxidase (AOX1) promoter. Three expression vectors were constructed and designated pCJ-βWT (wild-type bovine β-casein gene), pCJ- β68 (substitution of Ser70 for Leu70), and pCJ-β6873 (Ser70-Ser71 for Leu70-Pro71). Bovine β-casein produced in yeast was found to contain a sugar moiety on Asn68 (N-linked glycosylated) when produced from a strain containing the pCJ- β6873 construct in its chromosome. N-Glycosylation of bovine β-casein at position 68 was completely inhibited in transformants carrying vector pCJ- β68 with the single substitution of Ser70 for Leu70. The concentration of bovine β-casein in this expression system was in the range of 0.7-1.0 g/L. Byung-Kwon Choi Dairy Science http://works.bepress.com/rjimenez/41 http://works.bepress.com/rjimenez/41 Fri, 23 May 2008 15:55:27 PDT Recombinant DNA technology offers numerous opportunities for engineering food proteins and for studying their structure-function relationship. As part of the study of structure-function of bovine caseins, it is necessary to produce mutant proteins in experimental amounts and correlate their new structure to their physiochemical characteristics. To this end, bovine β-casein was expressed in the yeast Saccharomyces cerevisia by a fusion to the HXK1 (hexokinase P1) gene. Casein was produced during late exponential/early stationary phase of growth on glucose as would be predicted for a gene under the control of the HXK1 promoter. Bovine β-casein was posttranslationally modified by yeast. Internal phosphorylated forms were observed as well as a high molecular weight form that appeared to be O-glycosylated and largely localized to the periplasmic space. Rafael Jiménez-Flores Dairy Science http://works.bepress.com/rjimenez/40 http://works.bepress.com/rjimenez/40 Fri, 23 May 2008 15:55:23 PDT Protein source and saponins have been shown to influence lipid metabolism; however, little is known regarding the chemical interactions between proteins and saponins or the impact of addition of dietary saponins to different proteins on cholesterolemia. In the present study, quillaja saponin was added to casein and to isolated soy protein (ISP) and the saponin-protein interaction was investigated by gel electrophoresis and fluoroscopy. The impact on cholesterol metabolism also was investigated in gerbils. Results from the interaction studies showed that high molecular weight complexes were formed gradually between saponins and caseins, with β-casein being most susceptible. The resulting complexes differed drastically in charge and molecular weight. In contrast, soy proteins formed insoluble aggregates during heating independently of the presence of added quillaja saponin. Data from the animal study showed that addition of saponin to ISP did not affect serum lipids, while addition of saponin to casein resulted in significant decreases of LDL cholesterol and LDL/HDL ratios, resulting in values similar to those of ISP-fed animals. These results indicate that the effect of saponins on serum lipid profiles is dependent on the source of dietary protein. This could be explained by the finding that quillaja saponin reacted differently with caseins as compared to soy protein isolate. Susan M. Potter Dairy Science http://works.bepress.com/rjimenez/42 http://works.bepress.com/rjimenez/42 Fri, 23 May 2008 15:55:18 PDT Genomic vectors containing mutant bovine β-casein with putative glycosylation sites were constructed to study the functional properties of glycosylated β-casein and its possible effects in milk. The mutation was performed by PCR-based site-directed mutagenesis. The tripeptide sequence, Asn-X-Ser, was generated between Asn68 and Asn73 in mature β-casein. The resulting β-casein mutants were designated pCJB68 and pCJB6873. pCJB68 carries a substitution of Ser70 for Leu70 (Asn68-Ser69-Ser70-Pro71), and pCJB6873 carries a substitution of Ser70-Ser71 for Leu70-Pro71 (Asn68-Ser69-Ser70-Ser71). The two mutated genomic constructs were placed under control of the bovine α-lactalbumin promoter, and lines of mice expressing the pCJB68 and pCJB6873 have been established. The milk from transgenic mice contained bovine β-casein at levels up to 2-3 mg/mL. N-Linked glycosylation of bovine β-casein in the pCJB6873 line was confirmed by peptide-N-glycosidase F treatment, but glycosylation of bovine β-casein did not occur in pCJB68 mice. In addition, mouse casein micelles containing glycosylated bovine β-casein showed the largest median diameter and rough outer surface, compared to normal mouse casein micelles and micelles from transgenic milk containing bovine β-casein. Byung-Kwon Choi Dairy Science http://works.bepress.com/rjimenez/50 http://works.bepress.com/rjimenez/50 Fri, 23 May 2008 15:54:47 PDT Post-translational glycosylation of bovine β -casein (L70S/P71S) that results in Asn68-linked glycan on the protein was obtained in up to 30% of total β-casein expressed in the methylotrophic yeast Pichia pastoris. Among the growth/induction media used, buffered minimal glycerol (BMG)/buffered minimal methanol (BMM) media were best for the production of glycosylated bovine β-casein, indicating pH-dependent glycosylation. Glycosylated bovine β-casein (L70S/P71S) expressed in P. pastoris was purified to homogeneity by the combination of ammonium sulfate fractionation, Concanavalin A-Sepharose affinity column, and Mono Q anion-exchange FPLC. The purified glycosylated bovine β -casein was specific only to Concanavalin A, and the oligosaccharide structure of glycosylated β-casein was of high-mannose type. Unlike the hyperglycosylation that occurred in yeast, the majority of bovine β-casein was not hyperglycosylated in P. pastoris, and its molecular weight was estimated to be 33.6 kDa. Glycosylated bovine β-casein was normally phosphorylated to the same degree as native bovine β-casein. Byung-Kwon Choi Dairy Science http://works.bepress.com/rjimenez/47 http://works.bepress.com/rjimenez/47 Fri, 23 May 2008 15:54:00 PDT Solutions of tryptic hydrolysate of bovine β-lactoglobulin were fractionated by liquid-phase IEF in a preparative Rotofor cell at constant power for 2 h without ampholytes in order to identify interactions between peptides. The 20 peptide fractions collected were analyzed by capillary electrophoresis and SDS-PAGE under native, denaturing, and reducing conditions. The hydrolysate was shown to be composed mainly of acidic peptides (p/ 2-5, 62%) of molecular mass below 6 kDa, and numerous disulfide bonds were detected. Purified peptides (β-LG 15-20, 71-75, 76-82, and 84-91) were also focused individually and in mixtures and matched to components of the IEF fractions obtained from the tryptic hydrolysate of β-LG. The separation of acidic (β-LG 84-91) and basic (β-LG 76-82) peptides was achieved by IEF, whereas uncharged peptides (β-LG 15-20 and 71-75) were poorly separated due to their low electrophoretic mobility. Because no peptide-peptide interaction could be identified by IEF fractionation, it is suggested that electrical fields may decrease electrostatic interactions between charged peptides. Paule Emilie Groleau Dairy Science http://works.bepress.com/rjimenez/43 http://works.bepress.com/rjimenez/43 Fri, 23 May 2008 15:53:24 PDT Heat treatment during manufacturing of milk powder is one of the most important tools for manipulation of its functional properties, and it is the basis of the classification of these proteins into low-, medium-, and high-heat types. Slight differences in the sequences of the major proteins in milk (genetic variants) seem to have also a significant effect in milk powder processing (U.S. patent). Therefore, the effects of high-temperature storage and heat treatment on skim milk of defined genetic variants of β-lactoglobulin (β-LG) were measured. The samples had 45% total solids, the temperature of aging was 50°C, and the heat treatment was 90°C for 10 min prior to evaporation. Measurements on shear rate and on apparent viscosity were determined for each sample. During storage of the concentrated milk, the apparent viscosity and yield values increased markedly, and the age-dependent increase in viscosity in heat-treated concentrated skim milks was much more pronounced than in those prepared from unheated skim milks. The increase in apparent viscosity and yield value with storage time was notably different for milks containing different genetic variants. Unheated concentrated milks containing the B variant of β-LG showed the most rapid increase in apparent viscosity with storage time, whereas the viscosity increase was slowest in the concentrate containing the A variant. In contrast, heat-treated concentrated milks containing the A variant of β-LG showed the most rapid increase in viscosity with storage time, whereas the viscosity increase was slowest in the concentrate containing the AB variant. The changes in apparent viscosity of concentrated milk were largely reversible under high shear during the early stages of storage, but samples stored for a long time showed irreversible changes in apparent viscosity. Particle size analysis confirmed irreversible aggregation and fusion of casein particles during storage. Annie Bienvenue Dairy Science http://works.bepress.com/rjimenez/46 http://works.bepress.com/rjimenez/46 Fri, 23 May 2008 15:52:49 PDT Heat treatment of milk causes the heat-denaturable whey proteins to aggregate with κ-casein (κ-CN) via thiol-disulfide bond interchange reactions. The particular disulfide bonds that are important in the aggregates are uncertain, although Cys121 of β-lactoglobulin (β-LG) has been implicated. The reaction at 60°C between β-LG A and an activated κ-CN formed small disulfide-bonded aggregates. The tryptic peptides from this model system included a peptide with a disulfide bond between a Cys residue in the triple-Cys peptide [β-LG(102-124)] and κ-CN Cys88 and others between κ-CN Cys88 or κ-CN Cys11 and β-LG Cys160. Only the latter two novel disulfide bonds were identified in heated (90°C/20 min) milk. Application of computational search tools, notably MS2Assign and SearchXLinks, to the mass spectrometry (MS) and collision-induced dissociation (CID)-MS data was very valuable for identifying possible disulfide-bonded peptides. In two instances, peptides with measured masses of 4275.07 and 2312.07 were tentatively assigned to β-LG(102-135): κ-CN(11-13) and β-LG A(61- 69): κ-CN(87s97), respectively. However, sequencing using the CID-MS data demonstrated that they were, in fact, β-LG(1-40) and β-LG(41-60), respectively. This study supports the notion that reversible intramolecular disulfide-bond interchange precedes the intermolecular interchange reactions. Edwin K. Lowe Dairy Science http://works.bepress.com/rjimenez/45 http://works.bepress.com/rjimenez/45 Fri, 23 May 2008 15:52:21 PDT Changes in the structure and chemistry of β-lactoglobulin (β-LG) play an important role in the processing and functionality of milk products. In model β-LG systems, there is evidence that the aggregates of heated β-LG are held together by a mixture of intermolecular non-covalent association and heat-induced non-native disulfide bonds. Although a number of non-native disulfide bonds have been identified, little is known about the initial inter- and intramolecular disulfide bond rearrangements that occur as a result of heating. These interchange reactions were explored by examining the products of heat treatment to determine the novel disulfide bonds that form in the heated β-LG aggregates. The native protein and heat-induced aggregates were hydrolyzed by trypsin, and the resulting peptides, before and after reduction with dithiothreitol, were separated by high-performance liquid chromatography and their identities confirmed by electrospray ionization mass spectrometry. Comparisons of these peptide patterns showed that some of the Cys160 was in the reduced form in heated β-LG aggregates, indicating that the Cys160-Cys66 disulfide bond had been broken during heating. This finding suggests that disulfide bond interchange reactions between β-LG non-native monomers, or polymers, and other proteins could occur largely via Cys160. Lawrence K. Creamer Dairy Science