Increased Protein Yield Research Articles

Effects of ultrasound extraction on the physicochemical and emulsifying properties of rice bran protein

Abstract In this study, rice bran protein was prepared by ultrasound-assisted extraction, and its physicochemical and emulsifying properties were also evaluated. Results demonstrated that a significant increase in protein yield was observed when ultrasound-assisted method was employed for extracting protein. Noticeably, obtained rice bran protein possessed excellent physicochemical properties, such as oil absorption capacity, protein solubility and foaming property. More hydrophobic groups were exposed in the process of ultrasound-assisted extraction, which led to the increase of surface hydrophobicity. More importantly, the ultrasound-assisted extraction could improve emulsifying properties of rice bran protein, and the emulsions prepared using protein samples exhibited the great stability. Besides, it was also found that emulsifying properties of protein samples presented a decrease trend with increasing ultrasound power and time. All in all, ultrasound-assisted extraction is a suitable alternative process for preparing rice bran protein.

A Modelling Framework Linking Resource-Based Stochastic Translation to the Optimal Design of Synthetic Constructs

Simple SummaryIn synthetic biology, it is commonplace to design and insert gene expression constructs into cells for the production of useful proteins. In order to maximise production yield, it is useful to predict the performance of these “engineered cells” in advance of conducting experiments. This is typically a complex task, which in recent years has motivated the use of “whole-cell models” (WCMs) that act as computational tools for predicting different aspects of cell growth. Many useful WCMs exist, however a common problem is their over-simplification of ribosome movement on mRNA transcripts during translation. WCMs typically don’t consider that, for constructs with inefficient (“slow”) codons, ribosomes can stall and form “traffic jams”, thereby becoming unavailable for translation of other proteins. To more accurately address these scenarios, we have built a computational framework that combines whole-cell modelling with a detailed account of ribosome movement on mRNA. We show how our framework can be used to link the modular design of a gene expression construct (via its promoter, ribosome binding site and codon composition) to protein yield during continuous cell culture, with a particular focus on how the optimal design can change over time in the presence or absence of “slow” codons.The effect of gene expression burden on engineered cells has motivated the use of “whole-cell models” (WCMs) that use shared cellular resources to predict how unnatural gene expression affects cell growth. A common problem with many WCMs is their inability to capture translation in sufficient detail to consider the impact of ribosomal queue formation on mRNA transcripts. To address this, we have built a “stochastic cell calculator” (StoCellAtor) that combines a modified TASEP with a stochastic implementation of an existing WCM. We show how our framework can be used to link a synthetic construct’s modular design (promoter, ribosome binding site (RBS) and codon composition) to protein yield during continuous culture, with a particular focus on the effects of low-efficiency codons and their impact on ribosomal queues. Through our analysis, we recover design principles previously established in our work on burden-sensing strategies, namely that changing promoter strength is often a more efficient way to increase protein yield than RBS strength. Importantly, however, we show how these design implications can change depending on both the duration of protein expression, and on the presence of ribosomal queues.

Transient expression of recombinant proteins in plants.

Transient protein expression in plant cells is less time consuming than the production of whole transgenic plants. For transient expression, agroinfiltration is a simple and effective method to deliver transgenes into plant cells. After an Agrobacterium infection, recombinant proteins can be produced in plant cells from 3 to 10days. To increase protein yield, a deconstructed viral vector has been used. This chapter provides a detailed description of the transient expression of recombinant proteins in a well-developed host strain of Nicotiana benthamiana. This study also describes the necessary steps for the extraction of soluble proteins from agroinfiltrated leaves.

Altering the ratio of dietary palmitic and oleic acids affects production responses during the immediate postpartum and carryover periods in dairy cows

The objectives of our study were to determine the effects of altering the dietary ratio of palmitic (C16:0) and oleic (cis-9 C18:1) acids on production and metabolic responses of early-lactation dairy cows during the immediate postpartum period and to evaluate carryover effects of the treatment diets early in lactation. Fifty-six multiparous cows were used in a randomized complete block design and randomly assigned to 1 of 4 treatments (14 cows per treatment) fed from 1 to 24 d in milk (DIM). The treatments were: (1) control (CON) diet not supplemented with fatty acids (FA); (2) diet supplemented with a FA blend containing 80% C16:0 and 10% cis-9 C18:1 (80:10); (3) diet supplemented with a FA blend containing 70% C16:0 and 20% cis-9 C18:1 (70:20); and (4) diet supplemented with a FA blend containing 60% C16:0 and 30% cis-9 C18:1 (60:30). The FA supplement blends were added at 1.5% of diet DM by replacing soyhulls in the CON diet. All cows were offered a common diet from d 25 to 63 postpartum (carryover period) to evaluate carryover effects. Three preplanned contrasts were used to compare treatment differences: CON versus FA-supplemented diets (80:10 + 70:20 + 60:30)/3; the linear effect of cis-9 C18:1 inclusion in diets; and the quadratic effect of cis-9 C18:1 inclusion in diets. During the treatment period, FA-supplemented diets increased milk yield, 3.5% fat-corrected milk (FCM), and energy-corrected milk (ECM) compared with CON. Compared with CON, FA-supplemented diets increased milk fat content, milk fat yield, yield of mixed FA, and tended to increase protein yield and lactose yield. Also, compared with CON, FA-supplemented diets tended to increase body condition score (BCS) change. A treatment by time interaction was observed for body weight (BW), due to 80:10 inducing a greater BW loss over time compared with other treatments. Increasing cis-9 C18:1 in FA treatments tended to linearly increase dry matter intake (DMI) but did not affect milk yield, 3.5% FCM, ECM, and the yields of milk fat, protein and lactose. Increasing cis-9 C18:1 in FA treatments linearly decreased milk fat content and milk lactose content. Also, increasing cis-9 C18:1 in FA treatments linearly decreased BW and BCS losses. During the carryover period, compared with CON, FA-supplemented diets tended to increase milk yield. Also, FA-supplemented diets increased 3.5% FCM, ECM, and milk fat yield, and tended to increase milk protein yield compared with CON. A treatment by time interaction was observed for BW due to 80:10 increasing BW over time compared with CON. Our results indicate that feeding FA supplements containing C16:0 and cis-9 C18:1 during the immediate postpartum period increased milk yield and ECM compared with a nonfat supplemented control diet. Increasing cis-9 C18:1 in the FA supplement increased DMI and reduced BW and BCS losses. Additionally, the fat-supplemented diets fed during the immediate postpartum period had a positive carryover effect during early lactation, when cows were fed a common diet.

Effects of diet fermentability and supplementation of 2-hydroxy-4-(methylthio)-butanoic acid and isoacids on milk fat depression: 1. Production, milk fatty acid profile, and nutrient digestibility

The objectives of this experiment were to determine the effects of increased diet fermentability and polyunsaturated fatty acids (FA) with or without supplemental 2-hydroxy-4-(methylthio)-butanoic acid (HMTBa), isoacids (IA; isobutyrate, 2-methylbutyrate, isovalerate, and valerate) or the combination of these on milk fat depression (MFD). Ten Holstein cows (194 ± 58 DIM, 691 ± 69 kg BW, 28 ± 5 kg milk yield) were used in a replicated 5 × 5 Latin square design. Treatments included a high-forage control diet (HF-C), a low-forage control diet (LF-C) causing MFD by increasing starch and decreasing neutral detergent fiber (NDF), the LF-C diet supplemented with HMTBa at 0.11% (28 g/d), the LF-C diet supplemented with IA at 0.24% of dietary dry matter (60 g/d), and the LF-C diet supplemented with HMTBa and IA. Preplanned contrasts were used to compare HF-C versus LF-C and to examine the main effects of HMTBa or IA and their interactions within the LF diets. Dry matter intake was greater for LF-C versus HF-C, but milk yield remained unchanged. The LF-C diet decreased milk fat yield (0.87 vs. 0.98 kg/d) but increased protein yield compared with HF-C. As a result, energy-corrected milk was lower (28.5 vs. 29.6 kg/d) for LF-C versus HF-C. Although the concentration of total de novo synthesized FA in milk fat was not affected, some short- and medium-chain FA were lower for LF-C versus HF-C, but the concentrations of C18 trans-10 isomers were not different. Total-tract NDF apparent digestibility was numerically lower (42.4 vs. 45.6%) for LF-C versus HF-C. As the main effects, the decrease in milk fat yield observed in LF-C was alleviated by supplementation of HMTBa through increasing milk yield without altering milk fat content and by IA through increasing milk fat content without altering milk yield so that HMTBa or IA, as the main effects, increased milk fat yield within the LF diets. However, interactions for milk fat yield and ECM were observed between HMTBa and IA, suggesting no additive effect when used in combination. Minimal changes were found on milk FA profile when HMTBa was provided. However, de novo synthesized FA increased for IA supplementation. We detected no main effect of HMTBa, IA, and interaction between those on total-tract NDF digestibility. In conclusion, the addition of HMTBa and IA to a low-forage and high-starch diet alleviated moderate MFD. Although the mechanism by which MFD was alleviated was different between HMTBa and IA, no additive effects of the combination were observed on milk fat yield and ECM.

Hypotonic osmotic shock treatment to enhance lipid and protein recoveries from concentrated saltwater Nannochloropsis slurries

This study reports the use of hypotonic osmotic shock as a treatment step to enhance the recoveries of biofuel-convertible lipids and proteins from lipid-rich saltwater Nannochloropsis gaditana (N. gaditana) slurries (biomass content = ~140 mg biomass / g slurry, total lipid content = ~600 mg lipid /g biomass). The osmotic shock was induced through repeated washing of microalgal slurries with multiple batches of fresh water. Subjecting the slurries to 2 stages of freshwater washing resulted in a measurable damage to cell membranes (the uptake of membrane permeability marker increased by 6 folds), a partial loss of cell viability (only 64% of available cells were recoverable), and a minor release of free protein (~2 wt% of available protein) from the biomass into the interstitial space of the slurries. Hypotonic osmotic shock was revealed to be ineffective in rupturing N. gaditana slurries (only 13 ± 9% of available cells were ruptured after 2-stage washing) and, as such, had a limited prospect as a stand-alone cell disruption technology for the saltwater strain.The washing treatment, however, was found to be able to weaken the structural integrity of N. gaditana slurries and enhance the performance of subsequent mechanical or chemical cell disruption technologies when installed as a preparatory step. Applying the washing treatment prior to high-pressure homogenisation (HPH) and low-solvent-to-biomass ratio hexane extraction (hexane : slurry = 1:1 w/w) for the recovery of biofuel-convertible lipids increased the extent of cell rupture from 28 ± 8 to 46 ± 19% of available cells and more than doubled neutral lipid yield from 25.1 ± 2.0 to 64.6 ± 4.9 wt% of available neutral lipid. Initial analysis revealed that the washing treatment had a minimal energy cost (~6% of the total energy expenditure of downstream processing) and that its integration into HPH + hexane lipid recovery led to a 2.5 fold increase in the energy output of the biomass. Partnering the washing treatment with NaOH hydrolysis increased protein yield from 6.7 ± 2.4 to 31.9 ± 10.7 wt% of available protein.

Influence of phosphorus and zinc nutrition on yield and nutrient uptake in wheat (Triticum aestivum) and their residual effect on greengram (Vigna radiata)

A field experiment was conducted during 2014-16 at farmers' field in Panwari village of Agra district (UP) to study the direct and residual effects of phosphorus and zinc levels on yield, quality and nutrient uptake in wheat (Triticum aestivum L.) - greengram (Vigna radiata L.) cropping system using treatment combination of phosphorus (0, 30, 60 and 90 kg P2O5/ha) and zinc (0, 2, 4 and 6 kg/ha). The results revealed that the application of 60 kg P2O5/ha to wheat crop recorded significantly highest plant height (102.7 cm) and test weight (43.9 g). Phosphorus application in preceding wheat crop at 90 kg P2O5/ ha was found to be best for growth and yield attributes of greengram. Wheat crop responded significantly up to 60 kg P2O5/ ha by producing 5.39 t grain and 7.59 t straw/ ha. The residual effect of P in greengram was significant at 90 kg P2O5/ ha and grain and straw yield increments were to the extent of 15.8 and 12.0 %, respectively. Wheat crop responded to zinc application and 4 kg Zn/ ha could be adjudged response level. The residual effect of 6 kg Zn/ ha on succeeding greengram crop produced 12.3 and 7.1% higher grain and straw yield over control. Protein content in wheat and succeeding green gram increased significantly with P and Zn application but P was more effective in improving protein content in both the crops. There was a significant increase in protein yield of wheat and greengram with both the elements. Application of P and zinc showed substantial increase in P and Zn uptake by wheat and succeeding greengram crop over control. Nitrogen uptake by both the crops also improved significantly with these elements. The residual available P and Zn status in post harvest soil improved with the application of respective elements. Thus, application of 60 kg P2O5/ha along with 6 kg Zn/ha in wheat were optimum doses for obtaining higher productivity, produce quality and residual P and Zn in soil under wheat-green gram cropping sequence.

Effect of supplementing palmitic acid and altering the dietary ratio of n-6:n-3 fatty acids in low-fibre diets on production responses of dairy cows.

Supplementing palmitic acid (C16 : 0) in combination with modifying the dietary n-6:n-3 fatty acid (FA) ratio may benefit energy metabolism and milk responses of dairy cows. Twelve Holstein cows (70 (sd 11) days in milk) were used in a replicated 4 × 4 Latin square and allocated to four low-fibre diets (18·5 % forage neutral-detergent fibre) supplemented with no FA (CON), or 2·4 % C16 : 0-enriched supplement (PAL), 2·4 % mixture (2:1) of C16 : 0 and n-6 FA (PW6), and mixture (2:1) of C16 : 0 and n-3 FA (PW3). The dietary ratio of n-6:n-3 was increased with PW6 (10:1) and decreased with PW3 (2·8:1), whereas PAL alone made no change in the ratio (about 7:1). Compared with CON, all FA-supplemented treatments increased milk yield. However, feed and energy intakes were higher in PAL than PW3 or PW6, resulting in greater feed efficiency for PW3 and PW6 than PAL. Dietary FA supplements decreased milk protein concentration but tended to increase protein yield. Compared with CON and FA mixtures, PAL increased milk fat content and tended to increase milk SFA and atherosclerotic index. The concentration of milk n-3 FA was similar between CON and PW3. Feeding PAL increased milk energy output and decreased energy partitioning towards body reserves (-4·2 %), while this measure was positive for other treatments. Blood TAG and NEFA concentrations, but not β-hydroxybutyrate, were increased by FA-supplemented treatments. Feeding C16 : 0 combined with either n-6 or n-3 FA enhanced feed efficiency, alleviated the negative impacts on body energy reserves, but lowering the dietary n-6:n-3 ratio improved the FA profile of milk.

SUMO-fusion and autoinduction-based combinatorial approach for enhanced production of bioactive human interleukin-24 in Escherichia coli.

High-level production of recombinant human interleukin-24 (IL-24), a multifunctional immunomodulatory cytokine, has been challenging due primarily to its aggregation as inclusion bodies in the bacterial host while persistent poor-expression in the insect/mammalian expression systems. The present study presents a robust, vector-host combination (pE-SUMO-IL24), auto-inducible medium (YNG/M9NG), and a simple purification scheme for soluble, bioactive, and cost-effective production of native-like IL-24 (nIL-24) in Escherichia coli. The final protein yield, following a three-step purification scheme (IMAC, SEC, dialysis), was 98 mg/L in shake-flask culture (with scale-up potential), which was several folds higher than reported earlier. In vitro cytotoxicity assays with HeLa and HCT116 cancer cell lines (performed using different concentrations of nIL-24) and the fluorescence activated cell sorting analysis (FACS) revealed a dose- and concentration-dependent increase in the population of pro-apoptotic cells with concomitant, statistically significant drop in the number of cells existent at Go/G1-, S-, and G2/M-phases (P < 0.002). The bioactive nIL-24, developed through this study, holds promise for use in further functional characterizations/applications. KEY POINTS: • Yeast SUMO fusion partner at N-terminus for improved solubility of an otherwise insoluble IL-24 in E. coli. • Enhanced cell densities with concomitant several-fold increase in protein yield by lactose-inducible media. • Improved inhibition of cervical and colorectal carcinomas by native-like nIL-24 compared with Met-containing IL. • Heterologous nIL-24 may enable better understanding of the functional intricacies linked up with its unique cancer-specific features. Graphical abstract.

The Role of Agrotechnical Factors in Shaping the Protein Yield of Maize (Zea mays L.)

The subject of the work was the evaluation of maize protein yield conditioned by various agrotechnical factors. The test results are derived from two different field experiments covering a total of seven years of observation. Protein yield was significantly dependent on changing weather conditions in the growing seasons. Protein yield was significantly lower in the dry and hot years. The sum of precipitation for the month of May was positively correlated with maize protein yield. However, there was no correlation between the average air temperature and the amount of protein yield. The sum of atmospheric precipitation for the entire growing season (IV–X) shaped maize protein yield in over 96%. A non-linear relationship of the 2nd degree was determined between these traits. Sowing maize into the soil cultivated traditionally (autumn tillage), “stay-green” type cultivars and row fertilization positively influenced maize protein yield. Initial fertilization (regardless of depth) with two-component NP fertilizer, in relation to broadcast fertilization, significantly increased maize protein yield from 11.4% to 18%, respectively. The type of nitrogen fertilizer did not differentiate the value of this trait. Pre-sowing nitrogen application was more effective compared to the top dressing, and significantly increased protein yield. Row fertilization with two-component NP fertilizer clearly affected the content protein in grain. The interdependence of genetic and agronomic progress is an important factor shaping the potential of maize protein yield and cannot be attributed to either breeding (cultivar) or agronomy.

Structure-based design of prefusion-stabilized SARS-CoV-2 spikes.

The COVID-19 pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S) protein, which is metastable and difficult to produce recombinantly. Here, we characterized 100 structure-guided spike designs and identified 26 individual substitutions that increased protein yields and stability. Testing combinations of beneficial substitutions resulted in the identification of HexaPro, a variant with six beneficial proline substitutions exhibiting ~10-fold higher expression than its parental construct and the ability to withstand heat stress, storage at room temperature, and three freeze-thaw cycles. A 3.2 Å-resolution cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for SARS-CoV-2.

Codon Usage and Splicing Jointly Influence mRNA Localization.

SummaryIn the human genome, most genes undergo splicing, and patterns of codon usage are splicing dependent: guanine and cytosine (GC) content is the highest within single-exon genes and within first exons of multi-exon genes. However, the effects of codon usage on gene expression are typically characterized in unspliced model genes. Here, we measured the effects of splicing on expression in a panel of synonymous reporter genes that varied in nucleotide composition. We found that high GC content increased protein yield, mRNA yield, cytoplasmic mRNA localization, and translation of unspliced reporters. Splicing did not affect the expression of GC-rich variants. However, splicing promoted the expression of AT-rich variants by increasing their steady-state protein and mRNA levels, in part through promoting cytoplasmic localization of mRNA. We propose that splicing promotes the nuclear export of AU-rich mRNAs and that codon- and splicing-dependent effects on expression are under evolutionary pressure in the human genome.

Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization

The expression of functional, folded, and isotopically enriched membrane proteins is an enduring bottleneck for nuclear magnetic resonance (NMR) studies. Indeed, historically, protein yield optimization has been insufficient to allow NMR analysis of many complex Eukaryotic membrane proteins. However, recent work has found that manipulation of plasmid codons improves the odds of successful NMR-friendly protein production. In the last decade, numerous studies showed that matching codon usage patterns in recombinant gene sequences to those in the native sequence is positively correlated with increased protein yield. This phenomenon, dubbed codon harmonization, may be a powerful tool in optimizing recombinant expression of difficult-to-produce membrane proteins for structural studies. Here, we apply this technique to an inward rectifier K+ Channel (Kir) 3.1-KirBac1.3 chimera. Kir3.1 falls within the G protein-coupled inward rectifier K+ (GIRK) channel family, thus NMR studies may inform on the nuances of GIRK gating action in the presence and absence of its G Protein, lipid, and small molecule ligands. In our hands, harmonized plasmids increase protein yield nearly two-fold compared to the traditional ‘fully codon optimized’ construct. We then employ a fluorescence-based functional assay and solid-state NMR correlation spectroscopy to show the final protein product is folded and functional.

Destruction of Polyelectrolyte Microcapsules Formed on CaCO3 Microparticles and the Release of a Protein Included by the Adsorption Method.

The degradation of polyelectrolyte microcapsules formed on protein-free CaCO3 particles consisting of polyallylamine (PAH) and polystyrene sulfonate (PSS) and the resulting yield of protein in the presence of various salts of different concentrations, as well as at two pH values, was studied by fluorescence spectroscopy; the protein was incorporated into prepared microcapsules by adsorption. It was found that a high concentration of sodium chloride (2 M) leads to considerable dissociation of PAH, which is apparently due to the loosening of polyelectrolytes under the action of ionic strength. At the same time, 0.2 M sodium chloride and ammonium sulfate of the same ionic strength (0.1 M) exert less influence on the amount of dissociated polymer. In the case of ammonium sulfate (0.1 M), the effect is due to the competitive binding of sulfate anions to the amino groups of the polyelectrolyte. However, unlike microcapsules formed on CaCO3 particles containing protein, the dissociation of polyelectrolyte from microcapsules formed on protein-free particles increased with increasing temperature. Apparently, a similar effect is associated with the absence of a distinct shell, which was observed on microcapsules formed on protein-containing CaCO3 particles. The high level of the presence of Fluorescein isothiocyanate (FITC)-labeled Bovine Serum Albumin (BSA) in the supernatant is explained by the large amount of electrostatically bound protein and the absence of a shell that prevents the release of the protein from the microcapsules. In 2 M NaCl, during the observation period, the amount of the released protein did not exceed 70% of the total protein content in the capsules, in control samples, this value does not exceed 8%, which indicates the predominantly electrostatic nature of protein retention in capsules formed on protein-free CaCO3 particles. The increase in protein yield and peeling of PAH with increasing pH is explained by the proximity of pH 7 to the point of charge exchange of the amino group of polyelectrolyte, as a result of the dissociation of the microcapsule.

Effect of Micronutrients Application and Spraying Yeast Extract on Yield and Yield Components of Wheat (Triticum aestivum L.)

A field experiment was conducted during winter season of 2018-2019, at Al-Qurna district (75 km north of Basrah city centre). The aim of experiment was studding the effect of five levels of micronutrients (MN), Fe, Zn, Mn, Cu, B, and Mo: 0, 2 and 4 kg. ha-1 (soil application), 500 and 1000 ppm (foliar application), and three levels of activated yeast extract (AYE): 0, 3 and 6 g. L-1, in the yield and qualitative characteristics of wheat. The experiment was carried out using randomized completely block design in a split arrangement with three replicates. The main plots contained fertilizer levels. While the sub-plot occupied by Yeast levels. The results showed that spraying 1000 ppm from the solution of MN significantly increase the number of spikes per m2 , the number of grains in the spike, grain yield and biological yield with an increase by 15.4%, 18%, 27.5 % and 35% respectively compared to the control treatment, while the concentration of 500 ppm significantly increase protein yield with an increase of 35% compared to the control treatment. The results also showed that spraying 6 g. L- 1 of AYE differ significantly and gave the highest values of spikes number per m2 , number of grains per spike , grain yield , biological yield and protein yield, with an increase by 5.84%, 7.1%, 11.4% , 11.1% and 10.9% respectively compared to the control treatment. The interaction between application of 4 kg. MN ha-1 and the level of 6 g. L-1 AYE gave a significant superiority in the number of spikes (333.9 spike m-2), while the spraying of 1000 ppm + 6 g. L-1 AYE gave the highest grain yield (8.891 ton. ha-1) and biological yield (31.827 ton. ha-1).

Improved recovery of protein from soy grit by enzyme-assisted alkaline extraction

Recovery of protein from soy grit, its functional properties and possibility for the reduction of time of conventional alkaline extraction by the assistance of enzymes were studied. Enzymatic treatment was performed by commercial preparations of cellulase (NS22086), xylanase (NS22083) and pectinase (Vinozym) (applied separately or in combination) as well as by commercial carbohydrases cocktail (Enzyme complex, NS22119). Three different extractions were investigated - alkaline (at pH 8 for 1 h, 2 h or 3 h), enzyme-assisted aqueous (at pH 5.5 for 3 h) and enzyme-assisted alkaline extractions (enzymatic extraction for 1 h followed by alkaline extraction for 1 h or 2 h) at 50 °C and solid:liquid ratio 1:10 (w/v). The highest enhancement of recovery of protein was achieved by pretreatment of soy grit with enzyme cocktails. Treatment with Enzyme complex followed by 1 h alkaline extraction increased protein yield for 21% compared to 2 h alkaline extraction. Treatment by combination of individual cellulase, xylanase and pectinase followed by 2 h alkaline extraction enhanced protein yield for 13% in comparison to 3 h alkaline extraction. So, reduced time of alkaline extraction was attained by the assistance of carbohydrases cocktails with even positive effect on protein yield. In addition, protein from enzyme-assisted alkaline extraction exhibited ameliorated solubility, emulsifying and whipping properties compared to alkaline extracted protein.

The Effects of Temperature and Culture Medium Composition on Protein Recombinant JSU-pET Expression

There are several factors influencing the protein expression. The study was aimed to increase the yield of protein recombinant JSU-pET by applying combination of temperature and medium culture composition. This research was composed in a 2x2 factorial design with two level temperatures of 37&lt;sup&gt;o&lt;/sup&gt;C and 25&lt;sup&gt;o&lt;/sup&gt;C and 2 medium compositions of LB and mLB (modified). &lt;em&gt;Escherichia coli&lt;/em&gt; bearing JSU-pET plasmid were grown in two applied media and incubated in two applied temperatures. The cells were harvested by centrifugation the pellets were then lysed. The lysed cells were centrifuged, the supernatant then purified by Ni-NTA Resin Agarose. The purified JSU protein recombinant was characterized and quantified by spectrophotometer. The Result showed that the JSU protein recombinant was successfully expressed on the right size (37kDa) in all treatments even though the JSU-pET showed sharper band in m LB at 37&lt;sup&gt;o&lt;/sup&gt;C. Statistically the protein yield was not significant different (P&amp;gt;0.05), however culture temperature of 25&lt;sup&gt;o&lt;/sup&gt;C showed higher yield (0.618±0.095 vs 0.704±0.094) than cultured at 37&lt;sup&gt;o&lt;/sup&gt;C (0.598±0.137 vs 0.553±0.041), respectively either cultured in LB or mLB. This finding suggests that a lower culture temperature could increase protein yield both either in LB or modified LB.

Aquatic weeds as novel protein sources: Alkaline extraction of tannin-rich Azolla

The aquatic weed Azolla is a potential protein crop due to its prolific growth and high protein content, supported entirely by nitrogen-fixing symbionts. Alkaline protein extraction at pH 8 followed by acid precipitation allowed recovery of 16–26% of the biomass nitrogen, while at pH 10.5 nitrogen recovery improved to 35–54%. This pH effect was typical of ferns of the family Salviniaceae, and may be explained by high concentrations of condensed tannins (CTs) in the biomass that precipitate protein at mild pH. Two approaches were tested to increase protein yield and reduce protein binding by CTs. Pre-extraction with aqueous acetone (70 v/v%) removed 76–85% of the CTs and subsequent alkaline extraction at pH 12.5 and 95 °C recovered 38% of the biomass nitrogen. Extraction with 1.5% of PEG as a CT-binding agent, also permitted to recover 38% of the nitrogen, under milder conditions of pH 8 and 45 °C.

Inhibition of Autolysosome Formation Improves rrhGAA Production Driven by RAmy3D Promoter in Transgenic Rice Cell Culture

Although plant cell cultures produce low yields of recombinant proteins compared to other production systems, a dramatic increase of the heterologous protein production in transgenic rice cells was achieved with the alpha-amylase isozyme 3D (RAmy3D) promoter system. However, this expression system has inherent limitations in that gene expression is initiated by sucrose/glucose deprivation, concurrently triggering starvation-derived autophagy and rapid cell death. Decreased viability and culture longevity subsequently prevent further increment of production. In this study, we introduced autophagy inducers and inhibitors in the rrhGAA-producing transgenic rice cell cultures in order to explore their effects on production controlled by the RAmy3D promoter. The autophagy inducers rapamycin and CCI-779 increased autophagosome and autolysosome while concanamycin A1 and bafilomycin A successfully decreased autolysosome. Interestingly, autophagy inhibitors improved viability, DCW loss, and rrhGAA production, while autophagy inducers deteriorated these profiles compared to the control. As the production conditions under the death phase may facilitate protein degradation, and subsequently exacerbate functional activity, the size variant distribution and enzyme activity of the purified rrhGAAs were evaluated. However, no significant difference in rrhGAA degradation as well as GAA activity was observed compared to the control condition, thus indicating that the autophagy regulation is an efficient approach to increase protein yield in rice cell culture system for rrhGAA production.

Optimizing the dynamics of protein expression

Heterologously expressed genes require adaptation to the host organism to ensure adequate levels of protein synthesis, which is typically approached by replacing codons by the target organism’s preferred codons. In view of frequently encountered suboptimal outcomes we introduce the codon-specific elongation model (COSEM) as an alternative concept. COSEM simulates ribosome dynamics during mRNA translation and informs about protein synthesis rates per mRNA in an organism- and context-dependent way. Protein synthesis rates from COSEM are integrated with further relevant covariates such as translation accuracy into a protein expression score that we use for codon optimization. The scoring algorithm further enables fine-tuning of protein expression including deoptimization and is implemented in the software OCTOPOS. The protein expression score produces competitive predictions on proteomic data from prokaryotic, eukaryotic, and human expression systems. In addition, we optimized and tested heterologous expression of manA and ova genes in Salmonella enterica serovar Typhimurium. Superiority over standard methodology was demonstrated by a threefold increase in protein yield compared to wildtype and commercially optimized sequences.