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Protein Yield (protein + yield)
Selected AbstractsEffect of Nitrogen Rate and Stubble Height on Dry Matter Yield, Crude Protein Content and Crude Protein Yield of a Sorghum,Sudangrass Hybrid[Sorghum bicolor (L.) Moench × Sorghum sudanense (Piper) Stapf.] in the Three-Cutting SystemJOURNAL OF AGRONOMY AND CROP SCIENCE, Issue 4 2003S. Iptas Abstract In this study, the effects of nitrogen (N) rate (60, 120, 180 and 240 kg N ha,1 applied in three equal dressings at seeding and after the first and second cuttings) and stubble height (7, 14 and 21 cm) on the dry matter (DM) yield, crude protein (CP) content, and CP yield of a sorghum,sudangrass hybrid [Sorghum bicolor (L.) Moench × Sorghum sudanense (Piper) Stapf., cv. Pioneer 988] in the three-cut system was investigated. The N rate had no significant effect in the first and third cuttings, but in the second cutting DM yields increased significantly with increase in N rate. The highest yield of 9.1 t ha,1 was obtained with 80 kg N ha,1 for the average of 2 years at the second cutting, but no significant difference was found among the 40, 60 and 80 kg N ha,1 rates. CP content and yield were not significantly affected by N rate at the first and third cuttings, but CP content and yield were significantly affected by application of N at the second cutting. Stubble height had a significant effect on CP content at the third cutting. However, it had no significant effect on CP content at the first and second cuttings. Stubble height had a significant effect on the CP yield at the first cutting, but no significant effect on CP yield at the second and third cuttings. [source] An efficient protein preparation for proteomic analysis of developing cotton fibers by 2-DEELECTROPHORESIS, Issue 22 2006Yuan Yao Abstract Preparation of high-quality proteins from cotton fiber tissues is difficult due to high endogenous levels of polysaccharides, polyphenols, and other interfering compounds. To establish a routine procedure for the application of proteomic analysis to cotton fiber tissues, a new protocol for protein extraction was developed by optimizing a phenol extraction method combined with methanol/ammonium acetate precipitation. The protein extraction for 2-DE was remarkably improved by the combination of chemically and physically modified processes including polyvinylpolypyrrolidone (PVPP) addition, acetone cleaning, and SDS replacement. The protocol gave a higher protein yield and vastly greater resolution and spot intensity. The efficiency of this protocol and its feasibility in fiber proteomic study were demonstrated by comparison of the cotton fiber proteomes at two growth stages. Furthermore, ten protein spots changed significantly were identified by MS/tandem MS and their potential relationships to fiber development were discussed. To the best of our knowledge, this is the first time that a protocol for protein extraction from cotton fiber tissues appears to give satisfactory and reproductive 2-D protein profiles. The protocol is expected to accelerate the process of the proteomic study of cotton fibers and also to be applicable to other recalcitrant plant tissues. [source] Effects of grass pasture and concentrate-based feeding systems for spring-calving dairy cows in early spring on performance during lactationGRASS & FORAGE SCIENCE, Issue 3 2005E. Kennedy Abstract The effect of offering a total mixed ration of silage and concentrate (proportionately 0·44 silage) system [indoor feeding system (IF)] was compared with grazing at a high daily herbage allowance with a low level of concentrate supplementation [early grazing system (EG)] in early spring on the performance of spring-calving dairy cows in Ireland. Sixty-four spring-calving Holstein,Friesian dairy cows (mean calving date, 2 February) were allocated to one of two systems between 16 February and 4 April 2004. An equal number of primiparous and multiparous cows were assigned to each system. The dairy cows on the IF system were housed for a 7-week period and offered a diet of 10·9 kg DM cow,1 d,1 (s.d. 2·3) of concentrate, the remainder of the diet was 8·6 kg DM cow,1 d,1 (s.d. 1·9) of grass silage. The dairy cows on the EG system were offered a mean daily herbage allowance of 15·1 kg DM cow,1 d,1 (s.d. 3·7) and were supplemented with 3·0 kg DM cow,1 d,1 (s.d. 1·0) of concentrate. There was no difference in milk yield between the two systems but the cows in the EG system had a higher milk protein concentration (2·9 g kg,1) and a higher milk protein yield than in the IF system. Milk fat concentration was higher for cows in the IF than EG system (3·0 g kg,1). There was no difference in total daily dry-matter intake between the systems, measured in week 6 of the study. Mean live weight of the cows in the IF system was greater than in the EG system. The results of the study suggest that a slightly greater performance can be achieved by a system offering a high daily herbage allowance to spring-calving dairy cows in early lactation compared with a system offering a total mixed ration containing a high proportion of concentrate with grass silage. [source] The effect of forage type and level of concentrate supplementation on the performance of spring-calving dairy cows in early lactationGRASS & FORAGE SCIENCE, Issue 3 2002P. Dillon Abstract In 1993 and 1994, 40 cows in early lactation in early spring were assigned randomly to four feeding treatments. One group of cows was kept indoors with access to grass silage ad libitum, plus 6 kg of concentrate daily. The other three groups had access to grass pasture (5,6 h per day in 1993 and 11,12 h per day in 1994) plus grass silage similar to that fed to the previous group while indoors plus 6, 4 or 2 kg of concentrate daily. The average daily allocations of herbage (> 3·5 cm) were 8·5 and 14·0 kg DM cow,1 day,1 in 1993 and 1994 respectively. The treatments were applied for 8 weeks (26 February to 23 April) in 1993, and 7 weeks (11 March to 29 April) in 1994. Cows with access to pasture had lower (P < 0·001) silage dry-matter (DM) intakes and higher (P < 0·001) total forage DM intakes in both years than those kept indoors. This resulted in significantly higher yields of milk, fat, protein and lactose. Similarly, milk protein concentration was higher (P < 0·05 in 1993; P < 0·001 in 1994). There was a significant linear increase in total DM intake in both years with increased concentrate supplementation. In 1993, there was a linear increase in milk (P < 0·01), fat (P < 0·01), protein (P < 0·001) and lactose (P < 0·01) yields with increased concentrate supplementation. In 1994, only milk protein yield (P < 0·05) was increased. Concentrate supplementation had no effect on milk composition or liveweight change. Cows with access to grazed grass had higher liveweight gains (P < 0·05) than those kept indoors in both years. In 1993, increasing the energy intake increased the processing qualities of the milk produced. The results showed that access to grass pasture resulted in higher milk production, in reduced silage requirement and in reduced level of concentrate supplementation required for a given level of milk production with spring-calving cows in early lactation compared with those kept indoors. [source] Characterisation of soybean glycinin and ,-conglycinin fractionated by using MgCl2 instead of CaCl2INTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 1 2010Chong Liu Summary A simple two step precipitation method was used to investigate the effect of MgCl2 instead of CaCl2 on fractionation of soybean glycinin and ,-conglycinin. Compositional and physicochemical properties of the resulting protein fractions were characterised. The optimised procedure, in terms of protein yield, purity, phytate content and physicochemical properties, was obtained when the addition of 5 mm MgCl2 was used. After application of 5 mm MgCl2, the phytate content of the glycinin-rich and ,-conglycinin-rich fractions was about 0.4% and 1.3%, respectively, but the addition of 5 mm CaCl2 increased the phytate content of the glycinin-rich fraction to 1.25% and decreased that of ,-conglycinin-rich fraction to 0.67%. Low phytate protein product was suitable for use in infant formula and acidic food. The solubility of the glycinin-rich fractions with MgCl2 was significantly higher than that with CaCl2 at pH < 4.5. Application of MgCl2 improved thermal stability of the ,-conglycinin-rich fraction. [source] Metabolic and luteal function in winter-calving Spanish beef cows as affected by calf management and breedJOURNAL OF ANIMAL PHYSIOLOGY AND NUTRITION, Issue 3 2010J. Álvarez-Rodríguez Summary This experiment aimed at evaluating the effect of calf management and breed on the metabolic and luteal function of post-partum beef cows fed at maintenance. Fifty multiparous cows, 22 Parda de Montaña (PA) and 28 Pirenaica (PI), were assigned to either suckling once-daily for 30 min (RESTR) or ad libitum (ADLIB) from the day after calving. Blood samples were collected to analyse metabolites [non-esterified fatty acids (NEFA), ,-hydroxybutyrate, total protein and urea)], insulin-like growth factor-I (IGF-I) and progesterone (P4) at different intervals. Cows from RESTR maintained their live-weight (LW) over the first 3 months post-partum, whereas ADLIB cows lost nearly 4% LW. Both genotypes showed similar LW gains during this period (p > 0.10). Calf daily gains were lower in RESTR than in ADLIB treatment (p < 0.05), but similar across breeds (p > 0.10). Milk and lactose production were lower in RESTR cows than in ADLIB (p < 0.05). Milk and protein yield were greater in PA than in PI breed (p < 0.05). Serum NEFA, total protein and urea were higher in PI cows suckling ADLIB than in the rest (p < 0.05). Cows from PI breed had greater NEFA values than PA ones on the first week post-partum (p < 0.001). Circulating IGF-I was not affected by suckling frequency, breed nor their interaction (p > 0.10). Suckling frequency, but not breed, affected the interval from calving to first ovulation (p < 0.001), being shorter in RESTR than in ADLIB cows. In conclusion, the ad libitum suckling practice improved cow milk yield and offspring gain compared to once-daily suckling for 30 min from the day after calving, at the expense of impairing the onset of cyclicity. The effect of calf management was confounded with breed on the studied blood biochemical constituents, but any of these metabolites influenced the role of endocrine IGF-I in these genotypes. [source] Effects of rumen-protected methionine in a low protein ration on metabolic traits and performance of early lactating cows as opposed to rations with elevated crude protein contentJOURNAL OF ANIMAL PHYSIOLOGY AND NUTRITION, Issue 5 2000T. F. Kröber Summary A 5-week experiment with 24 multiparous early lactating Brown Swiss cows was conducted to investigate the effects of supplementary rumen-protected methionine in conjunction with dietary protein reduction on metabolism and performance after 1 week of control measurement. Three rations containing 175, 150 and 125 g of crude protein/kg feed dry matter were supplemented with methionine. The fourth ration, also only containing 125 g of crude protein/kg dry matter, remained unsupplemented. The four treatment groups had a similar metabolic supply of other essential amino acids, protein and energy, as calculated by various approaches. The two low protein rations were, however, slightly deficient in ruminally degraded protein. Treatment effects remained low on feed intake, forage meal pattern, milk yield and fat as well as lactose content. In contrast, the content and yield of milk protein significantly declined only in the unsupplemented low protein ration relative to the initial value. Compared with this ration, the decline in milk protein yield was clearly delayed in the supplemented low protein ration. Blood plasma methionine tended to be reduced without supplementation and to be increased with additional methionine. Supplementation of methionine reduced other plasma amino acids. Plasma insulin, glucose, lactate, ketone bodies and aspartate amino transferase activity indicated a certain liver stress and a somewhat elevated energy requirement with high and particularly with low protein content (when unsupplemented). Methionine improved metabolic protein utilization, followed by the lowest plasma, urine and milk urea levels in the supplemented low protein diet. In conclusion, no major adverse effects were assessed under the conditions tested. Supplementation of methionine may nevertheless be useful in rations with particularly low protein content fed to early lactating cows in order to prevent negative long-term effects which were only visible here as trends. Zusammenfassung Auswirkungen von pansengeschütztem Methionin in einer Niedrigproteinration im Vergleich zu Rationen mit erhöhtem Rohproteingehalt auf Stoffwechselmerkmale und Leistung von frischlaktierenden Milchkühen In einem fünfwöchigen Experiment mit 24 frischlaktierenden Braunviehkühen wurden die Auswirkungen einer Ergänzung mit pansengeschütztem Methionin bei gleichzeitiger Reduktion der Proteinzufuhr nach einer einwöchigen Kontrollphase geprüft. Drei Rationen mit 175, 150 und 125 g Rohprotein/kg T wurden mit Methionin ergänzt. Eine weitere Variante, ebenfalls nur mit 125 g Rohprotein/kg T, wurde nicht supplementiert. Die vier Varianten stellten gemäß verschiedener Futterbewertungsysteme eine vergleichbare metabolische Versorgung mit den übrigen essentiellen Aminosäuren, Protein und Energie sicher. Die Niedrigproteinvarianten enthielten allerdings etwas zu wenig pansenabbaubares Protein. Futteraufnahme, Muster des Grundfutterverzehrs, Milchleistung sowie Fett-und Laktosegehalt der Milch zeigten nur geringe Reaktion auf die Behandlungen. Milchproteingehalt und -menge waren nur in der nicht ergänzten Niedrigproteinvariante relativ zum Ausgangswert signifikant verringert. Im Vergleich zur unsupplementierten Niedrigproteinration war dagegen der Abfall mit Ergänzung deutlich verzögert. Gegenüber dem Ausgangswert war die Methioninkonzentration im Blutplasma ohne Ergänzung tendenziell erniedrigt, mit Ergänzung erhöht. Es erfolgte eine Verringerung der Plasmakonzentration anderer Aminosäuren durch die Methioninergänzung der Niedrigproteinration. Die Plasmaniveaus von Insulin, Glucose, Laktat, Ketonkörpern und Aspartataminotransferase-Aktivität lassen auf eine gewisse Leberbelastung und einen etwas höheren Energiebedarf mit hohem und besonders mit niedrigem Proteingehalt (unsupplementiert) schließen. Die Zulage an Methionin verbesserte die metabolische Proteinverwertung, so dass die Harnstoffgehalte in Blut, Milch und Harn in dieser Niedrigproteinvariante am niedrigsten waren. Insgesamt ergaben sich keine grösseren ungünstigen Effekte unter den getesteten Bedingungen. Dennoch könnte die Ergänzung von Rationen mit besonders niedrigem Rohproteingehalt mit Methionin beim Einsatz an frischlaktierende Kühe hilfreich sein, um negative Langzeitwirkungen zu verhindern, die sich hier lediglich andeuteten. [source] Proteomic analysis of RCL2 paraffin-embedded tissuesJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 5b 2008V. Belief Abstract Histopathological diagnosis in most of the world's hospitals is based upon formalin-fixed and paraffin-embedded (FFPE) tissues. Although this standard fixation and embedding procedure keeps the tissue in excellent form for morphological and immunohistological analysis, FFPE is inappropriate for nucleic acids and protein studies. We investigated the potential value of RCL2, a new non-toxic fixative, for sparing proteins preserved in paraffin-embedded tissues. Normal colonic mucosa tissue was fixed in RCL2 prior to paraffin embedding (RCL2P), and then processed for quality and quantity of protein conservation, as compared to frozen and FFPE tissues using complementary proteomic analysis approaches. Using 4 different protein extraction protocols, RCL2P tissue consistently showed the highest protein yield. Similar protein patterns were observed with RCL2P and frozen tissues using mono and bi-dimensional electrophoresis. Moreover, membrane, cytoplasmic and nuclear proteins, as well as phosphorylated proteins, were successfully detected using western-blot. Furthermore, protein patterns observed by mass spectrometry analysis after laser-captured microdissection were found to be identical for frozen and RCL2-fixed tissues. At last, immunohistochemistry using various antibodies showed comparable results between both tissue storage methods. We concluded that RCL2 has great potential for performing both morphological and molecular analyses on the same archival paraffin-embedded tissue sample, and can be a new method for investigating protein biomarkers. [source] PREPARATION AND CHARACTERIZATION OF MODIFIED WHEAT GLUTEN BY ENZYMATIC HYDROLYSIS-ULTRAFILTRATIONJOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2008JIN-SHUI WANG ABSTRACT The present work was aimed to investigate the characteristics of the modified wheat gluten by enzymatic hydrolysis followed ultrafiltration membrane fractionation. An 8% aqueous dispersion of wheat gluten was hydrolyzed by papain, and then subjected to ultrafiltration membrane for fractionation. The molecular mass of the peptides released during enzymatic hydrolysis of gluten was in the range of 5,000,15,000 D according to gel permeation chromatography profiles. The resultant hydrolysate (retentate fraction) after membrane fractionation produced the desired products with a protein yield of about 89%. The modified glutens had higher surface hydrophobicity compared with the original gluten. Moreover, the modified gluten protein had superior solubility to the original gluten in pH 3,10. The enhanced foaming capacity in the pH ranges studied had been found in the modified gluten. The improved functional properties of the modified gluten were related to the well-balanced distribution of hydrophilic and hydrophobic domains. The modified gluten was rich in glutamic acid and proline. PRACTICAL APPLICATIONS Gluten is an economically important byproduct during processing of wheat starch. The expanded utilization of gluten in food and nonfood applications had been limited for lack of desirable functional properties. To extend the range of available functional properties it needs to be investigated further. The limited enzymatic hydrolysis and membrane ultrafiltration had been extensively studied and had been shown to be very effective for improving the functional properties of the proteins. In this present study, wheat gluten was modified by enzymatic hydrolysis , membrane ultrafiltration fractionation. This method was proved to be an efficient way to modify wheat gluten protein. The functional properties of the modified glutens were significantly improved compared to the original gluten. It is expected that the modified gluten with enhanced functional properties could be used in a wide range of foods as new components to enhance their nutritional value and functional properties in food processing. [source] Effects of NaCl Concentration on Salting-in and Dilution During Salting-out on Soy Protein FractionationJOURNAL OF FOOD SCIENCE, Issue 4 2006N. A. Deak ABSTRACT:, Glycinin and ,-conglycinin are the main storage proteins in soybeans that can be fractionated by using alkali extraction, SO2, salting-in with NaCl, salting-out by dilution and pH adjustment to produce a glycinin-rich fraction, a ,-conglycinin,rich fraction, and an intermediate fraction, which is a mixture of the two proteins. Two different strategies were employed to optimize the procedure to achieve high efficiency in recovering the ,-conglycinin,rich fraction. The first strategy was to optimize salting-in effects of NaCl, and the effects of NaCl concentration on the yields and purities of the protein fractions were investigated. The maximum protein yield of the ,-conglycinin,rich fraction was obtained at 500 mM NaCl, but at the expense of purity. The optimum NaCl concentration was 250 mM, at which good protein yield (18.5%) and purity (84.5%) were achieved. At higher NaCl concentrations, the protein yields of the intermediate fractions were significantly lower, and the protein loss in the whey fraction increased. The second strategy was to improve the salting-out step for the ,-conglycinin,rich fraction. At 0- and 0.5-fold dilution, the purities and yields of the ,-conglycinin,rich fractions were significantly lower than at 1.0- and 2.0-fold dilution. There were no differences in protein yields or purities when using 1.0- or 2.0-fold dilution. According to these results, the recommended NaCl concentration for the salting-in step is 250 mM and the dilution factor for salting-out is 1.0. [source] Confirmation and refinement of a QTL on BTA5 affecting milk production traits in the Fleckvieh dual purpose cattle breedANIMAL GENETICS, Issue 1 2010A. Awad Summary We analysed a QTL affecting milk yield (MY), milk protein yield (PY) and milk fat yield (FY) in the dual purpose cattle breed Fleckvieh on BTA5. Twenty-six microsatellite markers covering 135 cM were selected to analyse nine half-sib families containing 605 sons in a granddaughter design. We thereby assigned two new markers to the public linkage map using the CRI-MAP program. Phenotypic records were daughter yield deviations (DYD) originating from the routinely performed genetic evaluations of breeding animals. To determine the position of the QTL, three different approaches were applied: interval mapping (IM), linkage analysis by variance component analysis (LAVC), and combined linkage disequilibrium (LD) and linkage (LDL) analysis. All three methods mapped the QTL in the same marker interval (BM2830-ETH152) with the greatest test-statistic value at 118, 119.33 and 119.33 cM respectively. The positive QTL allele simultaneously increases DYD in the first lactation by 272 kg milk, 7.1 kg milk protein and 7.0 kg milk fat. Although the mapping accuracy and the significance of a QTL effect increased from IM over LAVC to LDL, the confidence interval was large (13, 20 and 24 cM for FY, MY and PY respectively) for the positional cloning of the causal gene. The estimated averages of pair wise marker LD with a distance <5 cM were low (0.107) and reflect the large effective population size of the Fleckvieh subpopulation analysed. This low level of LD suggests a need for increase in marker density in following fine mapping steps. [source] Effects of DGAT1 and GHR on milk yield and milk composition in the Chinese dairy populationANIMAL GENETICS, Issue 6 2009D. Sun Summary Previous studies have demonstrated that the p.Lys232Ala substitution in the acylCoA: diacylglycerol acyltransferase (DGAT1) gene and the p.Phe279Tyr mutation in the growth hormone receptor (GHR) gene are the causative quantitative trait loci underlying milk yield and composition on BTA14 and BTA20 respectively. To examine their applications in the genetic improvement of Chinese dairy cattle productivity, we herein investigated the effects of the DGAT1 p.Lys232Ala and GHR p.Phe279Tyr mutations on milk, fat and protein yield, as well as fat and protein percentage in the milk of 1222 Holstein cows. Genotyping was performed using PCR-RFLP for DGAT1 or primer-introduced restriction analysis (PCR-PIRA) for GHR. With a mixed animal model, the significant associations of the DGAT1 p.Lys232Ala substitution with 305-day milk, fat and protein yield were identified (P = 0.0001). The DGAT1 allele that encode lysine at position 232 was associated with increased 305-day milk fat yield, but with decreased 305-day milk and protein yield, whereas the GHR p.Phe279Tyr mutation was found to be significantly associated with protein percentage (P = 0.0014). The allele substitution effect of p.279Phe by p.279Tyr may lead to a significant increase in protein percentage. Our findings indicate that DGAT1 p.232Ala and GHR p.279Phe could be used to increase milk yield and protein yield of Chinese Holstein cows. [source] Characterization of a QTL region affecting clinical mastitis and protein yield on BTA6ANIMAL GENETICS, Issue 5 2009H. Nilsen Summary Quantitative trait loci affecting clinical mastitis were detected and fine mapped to a narrow region on bovine chromosome 6 in the Norwegian Red cattle population. The region includes the casein gene cluster and several candidate genes thought to influence clinical mastitis. The most significant results were found for SNPs within the Mucin 7 gene. This gene encodes an antimicrobial peptide and constitutes part of the first line of defence for the mucosal immune system. Detection of long haplotypes extending several Mb may indicate that artificial selection has influenced the haplotype structures in the region. A search for selection sweeps supports this observation and coincides with association results found both by single SNP and haplotype analyses. Our analyses identified haplotypes carrying quantitative trait loci alleles associated with high protein yield and simultaneously fewer incidences of clinical mastitis. The fact that such haplotypes are found in relative high frequencies in Norwegian Red may reflect the combined breeding goal that is characterized by selection for both milk production and disease resistance. The identification of these haplotypes raises the possibility of overcoming the unfavourable genetic correlation between these traits through haplotype-assisted selection. [source] Identification of single nucleotide polymorphisms in the bovine CCL2, IL8, CCR2 and IL8RA genes and their association with health and production in Canadian HolsteinsANIMAL GENETICS, Issue 3 2007I. Leyva-Baca Summary The aim of this study was to identify the presence of SNPs in the chemokine genes CCL2 and IL8 and the chemokine receptor genes IL8RA and CCR2, and assess their potential contribution to variation in estimated breeding values (EBVs) for somatic cell score (SCS) and four other traits in Canadian Holstein bulls. Pools of DNA for bulls with high (H) and low (L) EBVs for SCS were used for identification of 11 SNPs. Two unreported SNPs were found in the CCL2 gene and one SNP was found in the CCR2 gene. Previously reported SNPs (three in the IL8 gene and five in the IL8RA chemokine receptor) were also identified. Two SNPs in CCL2, three in IL8, one in IL8RA and one in CCR2 were genotyped in Canadian Holstein bulls (n = 338) using tetra primer ARMS-PCR. We investigated associations of these seven polymorphisms with three production traits (milk yield, fat yield and protein yield) and one conformation trait related to mastitis (udder depth). The allele substitution effect for the CCL2 rs41255713:T>C SNP was significant at an experimental-wise level for milk yield (247.5 ± 79.9 kg) and protein yield (7.4 ± 2.3 kg) EBVs (P , 0.05). The associations of the SNPs with SCS EBVs were not significant at an experimental-wise level. However, the allele substitution effect of the CCR2 rs41257559:C>T SNP on SCS was significant at the comparison-wise level (,0.04 ± 0.02, P = 0.05), which might indicate a possible association in support of other published studies. Lastly, we assigned CCR2 to BTA22q24, where a previously QTL for SCS was identified. [source] Recombinant protein expression and solubility screening in Escherichia coli: a comparative studyACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2006Nick S. Berrow Producing soluble proteins in Escherichia coli is still a major bottleneck for structural proteomics. Therefore, screening for soluble expression on a small scale is an attractive way of identifying constructs that are likely to be amenable to structural analysis. A variety of expression-screening methods have been developed within the Structural Proteomics In Europe (SPINE) consortium and to assist the further refinement of such approaches, eight laboratories participating in the network have benchmarked their protocols. For this study, the solubility profiles of a common set of 96 His6 -tagged proteins were assessed by expression screening in E. coli. The level of soluble expression for each target was scored according to estimated protein yield. By reference to a subset of the proteins, it is demonstrated that the small-scale result can provide a useful indicator of the amount of soluble protein likely to be produced on a large scale (i.e. sufficient for structural studies). In general, there was agreement between the different groups as to which targets were not soluble and which were the most soluble. However, for a large number of the targets there were wide discrepancies in the results reported from the different screening methods, which is correlated with variations in the procedures and the range of parameters explored. Given finite resources, it appears that the question of how to most effectively explore `expression space' is similar to several other multi-parameter problems faced by crystallographers, such as crystallization. [source] Framework for the Rapid Optimization of Soluble Protein Expression in Escherichia coli Combining Microscale Experiments and Statistical Experimental DesignBIOTECHNOLOGY PROGRESS, Issue 4 2007R. S. Islam A major bottleneck in drug discovery is the production of soluble human recombinant protein in sufficient quantities for analysis. This problem is compounded by the complex relationship between protein yield and the large number of variables which affect it. Here, we describe a generic framework for the rapid identification and optimization of factors affecting soluble protein yield in microwell plate fermentations as a prelude to the predictive and reliable scaleup of optimized culture conditions. Recombinant expression of firefly luciferase in Escherichia coli was used as a model system. Two rounds of statistical design of experiments (DoE) were employed to first screen (D-optimal design) and then optimize (central composite face design) the yield of soluble protein. Biological variables from the initial screening experiments included medium type and growth and induction conditions. To provide insight into the impact of the engineering environment on cell growth and expression, plate geometry, shaking speed, and liquid fill volume were included as factors since these strongly influence oxygen transfer into the wells. Compared to standard reference conditions, both the screening and optimization designs gave up to 3-fold increases in the soluble protein yield, i.e., a 9-fold increase overall. In general the highest protein yields were obtained when cells were induced at a relatively low biomass concentration and then allowed to grow slowly up to a high final biomass concentration, >8 g·L,1. Consideration and analysis of the model results showed 6 of the original 10 variables to be important at the screening stage and 3 after optimization. The latter included the microwell plate shaking speeds pre- and postinduction, indicating the importance of oxygen transfer into the microwells and identifying this as a critical parameter for subsequent scale translation studies. The optimization process, also known as response surface methodology (RSM), predicted there to be a distinct optimum set of conditions for protein expression which could be verified experimentally. This work provides a generic approach to protein expression optimization in which both biological and engineering variables are investigated from the initial screening stage. The application of DoE reduces the total number of experiments needed to be performed, while experimentation at the microwell scale increases experimental throughput and reduces cost. [source] Optimization of the Human Adenosine A2a Receptor Yields in Saccharomyces cerevisiaeBIOTECHNOLOGY PROGRESS, Issue 5 2006Alison Wedekind G-protein coupled receptors (GPCRs) have been implicated in many human diseases and have emerged as important drug targets. Despite their medical relevance, knowledge about GPCR structure is limited, mainly due to difficulties associated with producing large amounts of functional protein and isolating this protein in functional form. However, our previous results indicate that when the human adenosine A2a receptor (A2aR) is expressed in Saccharomyces cerevisiae, high yields can be achieved. In light of these initial results and in anticipation of future purification efforts, experiments were conducted to optimize the system for maximum total protein yield. Emphasis was placed on not only producing large quantities of A2aR in each cell but also achieving high cell density in batch culture. Therefore, temperature, media pH, inducer concentration in the media, and induction cell density were tested for their effects on both cell growth (as measured by optical density, OD600) and per cell A2aR expression levels. For these studies, the A2aR expression levels were determined using a previously described A2aR-green fluorescent protein (GFP) fusion, so that expression could be monitored by fluorescence. Overall the data indicate that at late times (,60 h of expression) approximately 75% higher total batch protein yields can be achieved using lower expression temperatures or 60% higher using elevated induction cell density. The highest yields correspond to approximately 28 mg per liter of culture of total A2aR. Amounts of functional receptor were shown to increase on a per cell basis by decreasing expression temperature up to 25 h of expression, but at late time points (,60 h) functional yields did not appreciably improve. When compared to other reports of GPCR expression in yeast it is clear that this system is among those producing the highest GPCR protein yields per culture both before and after optimization. [source] Selection of locations of knots for linear splines in random regression test-day modelsJOURNAL OF ANIMAL BREEDING AND GENETICS, Issue 2 2010J. Jamrozik Summary Using spline functions (segmented polynomials) in regression models requires the knowledge of the location of the knots. Knots are the points at which independent linear segments are connected. Optimal positions of knots for linear splines of different orders were determined in this study for different scenarios, using existing estimates of covariance functions and an optimization algorithm. The traits considered were test-day milk, fat and protein yields, and somatic cell score (SCS) in the first three lactations of Canadian Holsteins. Two ranges of days in milk (from 5 to 305 and from 5 to 365) were taken into account. In addition, four different populations of Holstein cows, from Australia, Canada, Italy and New Zealand, were examined with respect to first lactation (305 days) milk only. The estimates of genetic and permanent environmental covariance functions were based on single- and multiple-trait test-day models, with Legendre polynomials of order 4 as random regressions. A differential evolution algorithm was applied to find the best location of knots for splines of orders 4 to 7 and the criterion for optimization was the goodness-of-fit of the spline covariance function. Results indicated that the optimal position of knots for linear splines differed between genetic and permanent environmental effects, as well as between traits and lactations. Different populations also exhibited different patterns of optimal knot locations. With linear splines, different positions of knots should therefore be used for different effects and traits in random regression test-day models when analysing milk production traits. [source] Comparison of estimated breeding values, daughter yield deviations and de-regressed proofs within a whole genome scan for QTLJOURNAL OF ANIMAL BREEDING AND GENETICS, Issue 6 2001H. Thomsen An important issue in quantitative trait loci (QTL) detection is the use of phenotypic measurement as a dependent variable. Daughter yield deviations (DYDs) as the unit of choice are not available for all traits of interest. The use of de-regressed proofs (DRPFs) of estimated breeding values (EBVs) is an alternative to using daughter yield deviations. The objective of this study was to examine possible differences between DYDs and DRPFs within the use of QTL detection. The pedigree used was part of the granddaughter design of the German QTL effort. Consisting marker maps for livestock species were derived from all available data of 16 German Holstein paternal half-sib families with a total of 872 sires. The number of progeny ranged from 19 to 127. A whole genome scan was performed using weighted and unweighted multimarker regression with DYDs, DRPFs and EBVs as dependent variables for the traits milk, fat and protein yields. Results were compared with respect to the number of QTL detected. A similar number of QTL was detected with DRPFs and DYDs. Also, when dependent variables were weighted according to the variance of the trait, a higher number of QTL was detected at the desired level of significance as compared to using unweighted variables. Vergleich von Zuchtwerten, Daughter Yield Deviation und deregressierten Zuchtwerten bei der Genomanalyse zum Nachweis für QTL Ein bedeutender Einflussfaktor in der QTL-Analyse (QTL: Genorte für quantitativemarkmale) ist die Wahl der abhängigen Leistungsvariablen. Da Daughter Yield Deviations (DYDs) aber nicht für jedes Leistungsmerkmal zur Verfügung stehen, sollte untersucht werden, ob die De-regression von Zuchtwerten als alternative Variable in der QTL Analyse verwendet werden kann. Für die Untersuchung wurde ein Teil des Tiermaterials verwendet, das im Rahmen des Genomanalyseprojektes der Arbeitsgemeinschaft Deutscher Rinderzüchter untersucht wurde. Es standen 872 Bullen aus 16 väterlichen Halbgeschwisterfamilien der Rasse Deutsche Holsteins zur Verfügung. Die Zahl der Nachkommen pro Familie variierte von 19 bis 127. Unter Verwendung eines gewichteten als auch ungewichteten Multi-Marker Regressionsansatzes wurde ein Genomscan für die Leistungsmerkmale Milch-kg, Fett-kg und Eiweiss-kg durchgeführt, wobei als abhängige Variablen Zuchtwerte (EBV), Daughter Yield Deviations (DYD) und de-regressierte Zuchtwerte (DRPF) verwendet wurden. Die De-regression wurde auf der Basis der effektiven Töchterzahl, der Heritabilität des Merkmals und der additiv genetischen Verwandtschaftsmatrix durchgeführt. Alle Ergebnisse wurden in Hinblick auf die Anzahl der entdeckten QTL verglichen. Es zeigten sich dabei keine wesentlichen Unterschiede bei der Verwendung von DYDs und DRPFs. Die Rate der QTL Entdeckungen war bei beiden verwendeten Zuchtwerten annähernd gleich. Über den Vergleich unterschiedlicher Leistungsvariablen hinaus wurden die Ergebnisse der ungewichteten und gewichteten Analyse einander gegenübergestellt. Es konnte gezeigt werden, dass bei einer Gewichtung des Merkmals die Rate der QTL-Entdeckungen bei einem bestimmten Signifikanzniveau deutlich höher ist. [source] Effects of NaCl Concentration on Salting-in and Dilution During Salting-out on Soy Protein FractionationJOURNAL OF FOOD SCIENCE, Issue 4 2006N. A. Deak ABSTRACT:, Glycinin and ,-conglycinin are the main storage proteins in soybeans that can be fractionated by using alkali extraction, SO2, salting-in with NaCl, salting-out by dilution and pH adjustment to produce a glycinin-rich fraction, a ,-conglycinin,rich fraction, and an intermediate fraction, which is a mixture of the two proteins. Two different strategies were employed to optimize the procedure to achieve high efficiency in recovering the ,-conglycinin,rich fraction. The first strategy was to optimize salting-in effects of NaCl, and the effects of NaCl concentration on the yields and purities of the protein fractions were investigated. The maximum protein yield of the ,-conglycinin,rich fraction was obtained at 500 mM NaCl, but at the expense of purity. The optimum NaCl concentration was 250 mM, at which good protein yield (18.5%) and purity (84.5%) were achieved. At higher NaCl concentrations, the protein yields of the intermediate fractions were significantly lower, and the protein loss in the whey fraction increased. The second strategy was to improve the salting-out step for the ,-conglycinin,rich fraction. At 0- and 0.5-fold dilution, the purities and yields of the ,-conglycinin,rich fractions were significantly lower than at 1.0- and 2.0-fold dilution. There were no differences in protein yields or purities when using 1.0- or 2.0-fold dilution. According to these results, the recommended NaCl concentration for the salting-in step is 250 mM and the dilution factor for salting-out is 1.0. [source] A modified tandem affinity purification strategy identifies cofactors of the Drosophila nuclear receptor,dHNF4PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2006Ping Yang Abstract With the completion of numerous genome projects, new high-throughput methods are required to ascribe gene function and interactions. A method proven successful in yeast for protein interaction studies is tandem affinity purification (TAP) of native protein complexes followed by MS. Here, we show that TAP, using Protein,A and CBP tags, is not generally suitable for the purification and identification of proteins from tissues. A head-to-head comparison of tags shows that two others, FLAG and His, provide protein yields from Drosophila tissues that are an order of magnitude higher than Protein,A and CBP. FLAG-His purification worked sufficiently well so that two cofactors of the Drosophila nuclear receptor protein,dHNF4 could be purified from whole animals. These proteins, Hsc70 and Hsp83, are important chaperones and cofactors of other nuclear receptor proteins. However, this is the first time that they have been shown to interact with a non-steroid binding nuclear receptor. We show that the two proteins increase the ability of dHNF4 to bind DNA in,vitro and to function in,vivo. The tags and approaches developed here will help facilitate the routine purification of proteins from complex cells, tissues and whole organisms. [source] Optimal nitrogen supply as a key to increased and sustained production of a monoclonal full-size antibody in BY-2 suspension culture,BIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010T. Holland Abstract Plant cell cultures have been used as expression hosts for recombinant proteins for over two decades. The quality of plant cell culture-produced proteins such as full-size monoclonal antibodies has been shown to be excellent in terms of protein folding and binding activity, but the productivity and yield fell short of what was achieved using mammalian cell culture, in which the key to gram-per-liter expression levels was strain selection and medium/process optimization. We carried out an extensive media analysis and optimization for the production of the full-size human anti-HIV antibody 2G12 in N. tabacum cv. BY-2. Nitrogen source and availability was found to be one key factor for the volumetric productivity of plant cell cultures. Increased amounts of nitrate in the culture medium had a dramatic impact on protein yields, resulting in a 10,20-fold increase in product accumulation through a combination of enhanced secretion and higher stability. The results were scalable from shake flasks to stirred-tank bioreactors, where the maximum yield per cultivation volume was 8,mg,L,1 over 7 days. During the stationary phase, antibody levels were 150-fold higher in nitrogen-enriched medium compared to standard medium. The enhanced medium appeared not to affect antibody quality and activity, as determined by Western blots, surface plasmon resonance binding assays and N -glycan analysis. Biotechnol. Bioeng. 2010;107: 278,289. © 2010 Wiley Periodicals, Inc. [source] High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acidsBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Aaron R. Goerke Abstract We describe an E. coli -based cell-free system for the production of proteins with a non-natural amino acid (nnAA) incorporated site-specifically (modified protein). The mutant Methanococcus jannaschii tyrosyl-tRNA synthetase (mTyrRS) and tRNATyr pair were used as orthogonal elements. The mTyrRS experienced proteolysis and modified protein yields improved with higher synthetase addition (200,300 µg/mL). Product yields were also improved by increasing levels of total protein to 20 mg protein/mL and available vesicle surface area to 0.5 m2/mL. This new E. coli -based cell-free procedure produced up to 400 µg/mL of eCAT109pAz, 660 µg/mL of eDHFR10pAz, and 210 µg/mL of mDHFR31pAz with p -azido- L -phenylalanine (pAz) incorporated site-specifically at the amber nonsense codon. O -methyl- L -tyrosine and p -acetyl- L -phenylalanine were incorporated by similar protocols. The desired specificity for incorporation of the nnAA by the cell-free system was confirmed. Additionally, the modified proteins were enzymatically active and reactive for copper(I)-catalyzed (3,+,2) cycloadditions (click chemistry). Biotechnol. Bioeng. 2009;102: 400,416. © 2008 Wiley Periodicals, Inc. [source] Protein recovery from enzyme-assisted aqueous extraction of soybeanBIOTECHNOLOGY PROGRESS, Issue 2 2010Kerry A. Campbell Abstract Enzyme-assisted aqueous oil extraction from soybean is a "green" alternative to hexane extraction that must realize potential revenues from a value-added protein co-product. Three technologies were investigated to recover protein from the skim fraction of an aqueous extraction process. Ultrafiltration achieved overall protein yields between 60% and 64%, with solids protein content of 70%, and was effective in reducing stachyose content, with fluxes between 4 and 10 L/m2 hr. Protein content was limited because of high retention of lipids and the loss of polypeptides below 13.6 kDa. Isoelectric precipitation was effective in recovering the minimally hydrolyzed proteins of skim, with a protein content of 70%, again limited by lipid content. However, protein recovery was only 30% because of the greater solubility of the hydrolyzed proteins. Recovery by the alternative of protein capture on dextran-grafted agarose quaternary-amine expanded bed adsorption resins decreased with decreasing polypeptide molecular weight. Proteins with molecular mass greater than 30 kDa exhibited slow adsorption rates. Expanded bed adsorption was most effective for recovery of proteins with molecular weight between 30 and 12 kDa. Overall, adsorption protein yields were between 14% and 17%. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Mcl-1 overexpression leads to higher viabilities and increased production of humanized monoclonal antibody in Chinese hamster ovary cellsBIOTECHNOLOGY PROGRESS, Issue 4 2009Brian S. Majors Abstract Bioreactor stresses, including nutrient deprivation, shear stress, and byproduct accumulation can cause apoptosis, leading to lower recombinant protein yields and increased costs in downstream processing. Although cell engineering strategies utilizing the overexpression of antiapoptotic Bcl-2 family proteins such as Bcl-2 and Bcl-xL potently inhibit apoptosis, no studies have examined the use of the Bcl-2 family protein, Mcl-1, in commercial mammalian cell culture processes. Here, we overexpress both the wild type Mcl-1 protein and a Mcl-1 mutant protein that is not degraded by the proteasome in a serum-free Chinese hamster ovary (CHO) cell line producing a therapeutic antibody. The expression of Mcl-1 led to increased viabilities in fed-batch culture, with cell lines expressing the Mcl-1 mutant maintaining ,90% viability after 14 days when compared with 65% for control cells. In addition to enhanced culture viability, Mcl-1-expressing cell lines were isolated that consistently showed increases in antibody production of 20,35% when compared with control cultures. The quality of the antibody product was not affected in the Mcl-1-expressing cell lines, and Mcl-1-expressing cells exhibited 3-fold lower caspase-3 activation when compared with the control cell lines. Altogether, the expression of Mcl-1 represents a promising alternative cell engineering strategy to delay apoptosis and increase recombinant protein production in CHO cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Framework for the Rapid Optimization of Soluble Protein Expression in Escherichia coli Combining Microscale Experiments and Statistical Experimental DesignBIOTECHNOLOGY PROGRESS, Issue 4 2007R. S. Islam A major bottleneck in drug discovery is the production of soluble human recombinant protein in sufficient quantities for analysis. This problem is compounded by the complex relationship between protein yield and the large number of variables which affect it. Here, we describe a generic framework for the rapid identification and optimization of factors affecting soluble protein yield in microwell plate fermentations as a prelude to the predictive and reliable scaleup of optimized culture conditions. Recombinant expression of firefly luciferase in Escherichia coli was used as a model system. Two rounds of statistical design of experiments (DoE) were employed to first screen (D-optimal design) and then optimize (central composite face design) the yield of soluble protein. Biological variables from the initial screening experiments included medium type and growth and induction conditions. To provide insight into the impact of the engineering environment on cell growth and expression, plate geometry, shaking speed, and liquid fill volume were included as factors since these strongly influence oxygen transfer into the wells. Compared to standard reference conditions, both the screening and optimization designs gave up to 3-fold increases in the soluble protein yield, i.e., a 9-fold increase overall. In general the highest protein yields were obtained when cells were induced at a relatively low biomass concentration and then allowed to grow slowly up to a high final biomass concentration, >8 g·L,1. Consideration and analysis of the model results showed 6 of the original 10 variables to be important at the screening stage and 3 after optimization. The latter included the microwell plate shaking speeds pre- and postinduction, indicating the importance of oxygen transfer into the microwells and identifying this as a critical parameter for subsequent scale translation studies. The optimization process, also known as response surface methodology (RSM), predicted there to be a distinct optimum set of conditions for protein expression which could be verified experimentally. This work provides a generic approach to protein expression optimization in which both biological and engineering variables are investigated from the initial screening stage. The application of DoE reduces the total number of experiments needed to be performed, while experimentation at the microwell scale increases experimental throughput and reduces cost. [source] Optimization of the Human Adenosine A2a Receptor Yields in Saccharomyces cerevisiaeBIOTECHNOLOGY PROGRESS, Issue 5 2006Alison Wedekind G-protein coupled receptors (GPCRs) have been implicated in many human diseases and have emerged as important drug targets. Despite their medical relevance, knowledge about GPCR structure is limited, mainly due to difficulties associated with producing large amounts of functional protein and isolating this protein in functional form. However, our previous results indicate that when the human adenosine A2a receptor (A2aR) is expressed in Saccharomyces cerevisiae, high yields can be achieved. In light of these initial results and in anticipation of future purification efforts, experiments were conducted to optimize the system for maximum total protein yield. Emphasis was placed on not only producing large quantities of A2aR in each cell but also achieving high cell density in batch culture. Therefore, temperature, media pH, inducer concentration in the media, and induction cell density were tested for their effects on both cell growth (as measured by optical density, OD600) and per cell A2aR expression levels. For these studies, the A2aR expression levels were determined using a previously described A2aR-green fluorescent protein (GFP) fusion, so that expression could be monitored by fluorescence. Overall the data indicate that at late times (,60 h of expression) approximately 75% higher total batch protein yields can be achieved using lower expression temperatures or 60% higher using elevated induction cell density. The highest yields correspond to approximately 28 mg per liter of culture of total A2aR. Amounts of functional receptor were shown to increase on a per cell basis by decreasing expression temperature up to 25 h of expression, but at late time points (,60 h) functional yields did not appreciably improve. When compared to other reports of GPCR expression in yeast it is clear that this system is among those producing the highest GPCR protein yields per culture both before and after optimization. [source] Enhanced Secretion of Heterologous Proteins in Pichia pastoris Following Overexpression of Saccharomyces cerevisiae Chaperone ProteinsBIOTECHNOLOGY PROGRESS, Issue 4 2006Wei Zhang In Pichia pastoris, secretory proteins are folded and assembled in the endoplasmic reticulum (ER). However, upon introduction of foreign proteins, heterologous proteins are often retained in the cytoplasm or in the ER as a result of suboptimal folding conditions, leading to protein aggregation. The Hsp70 and Hsp40 chaperone families in the cytoplasm or in ER importantly regulate the folding and secretion of heterologous proteins. However, it is not clear which single chaperone is most important or which combination optimally cooperates in this process. In the present study we evaluated the role of the chaperones Kar2p, Sec63, YDJ1p, Ssa1p, and PDI from Saccharomyces cerevisiae. We found that the introduction of Kar2p, Ssa1p, or PDI improves protein secretion 4,7 times. In addition, we found that the combination chaperones of YDJ1p/PDI, YDJ1p/Sec63, and Kar2p/PDI synergistically increase secretion levels 8.7, 7.6, and 6.5 times, respectively. Therefore, additional integration of chaperone genes can improve the secretory expression of the heterologous protein. Western blot experiments revealed that the chaperones partly relieved the secretion bottleneck resulting from foreign protein introduction in P. pastoris. Therefore, the findings from the present study demonstrate the presence of a network of chaperones in vivo, which may act synergistically to increase recombinant protein yields. [source] | |||||||||||||||||||