Yield Of Soluble Protein Research Articles

Enhancing co-translational folding of heterologous protein by deleting non-essential ribosomal proteins in Pichia pastoris

BackgroundTranslational regulation played an important role in the correct folding of heterologous proteins to form bioactive conformations during biogenesis. Translational pausing coordinates protein translation and co-translational folding. Decelerating translation elongation speed has been shown to improve the soluble protein yield when expressing heterologous proteins in industrial expression hosts. However, rational redesign of translational pausing via synonymous mutations may not be feasible in many cases. Our goal was to develop a general and convenient strategy to improve heterologous protein synthesis in Pichia pastoris without mutating the expressed genes.ResultsHere, a large-scale deletion library of ribosomal protein (RP) genes was constructed for heterologous protein expression in Pichia pastoris, and 59% (16/27) RP deletants have significantly increased heterologous protein yield. This is due to the delay of 60S subunit assembly by deleting non-essential ribosomal protein genes or 60S subunit processing factors, thus globally decreased the translation elongation speed and improved the co-translational folding, without perturbing the relative transcription level and translation initiation.ConclusionGlobal decrease in the translation elongation speed by RP deletion enhanced co-translational folding efficiency of nascent chains and decreased protein aggregates to improve heterologous protein yield. A potential expression platform for efficient pharmaceutical proteins and industrial enzymes production was provided without synonymous mutation.

Enhancement of soluble protein, polypeptide production and functional properties of heat-denatured soybean meal by fermentation of Monascus purpureus 04093

ABSTRACTThe aim of this study was to investigate the effect of fermentation parameters on heat-denatured soybean meal by Monascus purpureus 04093 on the soluble protein and polypeptide production and to evaluate the antioxidant activity and functional properties of the fermented products. The optimal fermentation parameters were obtained as follows: 11.25% soybean meal loaded in a 80 g/500 mL flask with 0.02 g/L Na2SO4 at 28°C and pH 6.5 for 96 h. The soluble protein and polypeptide yield reached 290.9 ± 3.4 mg/g and 106.6 ± 0.9 mg/g, respectively, which was 3.25 and 12.25 times that of the control, respectively. Moreover, the polysaccharide and amino acid contents, the antioxidant activities, the emulsifying properties and foaming capacities were significantly enhanced by Monascus fermentation (P < 0.05). The results indicated the viability of soybean meal in combination with Monascus for producing proteins with desirable characteristics as functional ingredients utilized in the food industry.

Efficient Keratinolysis of Poultry Feather Waste by the Halotolerant Keratinase from Salicola Marasensis.

Sustainable development in the bio-treatment of large-scale biomass bulks requires high performance enzymes adapted to extreme conditions. An extracellular keratinolytic extract was obtained from the culture broth of a halotolerant strain of Salicola marasensis. Keratin hydrolyzing activity of the concentrated enzyme extract was observed on a 100 mg of pretreated feather waste. The concentrated enzyme was able to hydrolyze the poultry feathers by 25% after 12 h incubation. The bio-waste material was optimally hydrolyzed at pH 9 and temperature of 40 °C. Among reductants, 1,4-dithiothreitol, L-cysteine, 2-mercaptoethanol, glutathione, and sodium sulfate showed the most remarkable effect on the bio-waste keratinolysis, while the tested surfactants and urea had no significant effect on the keratinolytic activity. Hexane and hexadecane indicated strong effect on keratinase activity and bio-treatment in the presence of 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) as a hydrophobic ionic liquid resulted in a maximal of 80% extraction yield of soluble proteins from feathers. Considering the stability of the extracellular keratinolytic content in [BMIM][PF6], the observed keratinase activity was noteworthy suggesting that the secreted enzyme may contribute to the bioconversion of feather wastes.

Effect of endogenous phenoloxidase on protein solubility and digestibility after processing of Tenebrio molitor, Alphitobius diaperinus and Hermetia illucens

Upon extracting soluble proteins from insects as potential food ingredient, endogenous enzymes, such as phenoloxidases, are expected to negatively affect protein properties. The effect of phenoloxidases on solubility and digestibility of proteins was investigated for larvae of Tenebrio molitor, Alphitobius diaperinus and Hermetia illucens. Phenoloxidase inhibition was done using blanching (50 s, 90 °C) before extraction or extracting in presence of sulfite.Similar soluble protein yields and compositions were found without and with sulfite addition, whereas blanching decreased soluble protein yield. Upon in-vitro hydrolysis by pepsin and trypsin, soluble proteins from H. illucens were more digestible than those of T. molitor and A. diaperinus. Phenoloxidase activity during grinding negatively affected in-vitro pepsin hydrolysis.Besides phenoloxidase activity, also endogenous proteases were shown to remain active at pH 8 in extracts containing sulfite and after blanching of larvae. This stresses that protease activity needs to be carefully controlled in the design of insect based ingredients.

Ancestral diterpene cyclases show increased thermostability and substrate acceptance.

Bacterial diterpene cyclases are receiving increasing attention in biocatalysis and synthetic biology for the sustainable generation of complex multicyclic building blocks. Herein, we explore the potential of ancestral sequence reconstruction (ASR) to generate remodeled cyclases with enhanced stability, activity, and promiscuity. Putative ancestors of spiroviolene synthase, a bacterial class I diterpene cyclase, display an increased yield of soluble protein of up to fourfold upon expression in the model organism Escherichia coli. Two of the resurrected enzymes, with an estimated age of approximately 1.7 million years, display an upward shift in thermostability of 7-13 °C. Ancestral spiroviolene synthases catalyze cyclization of the natural C20 -substrate geranylgeranyl diphosphate (GGPP) and also accept C15 farnesyl diphosphate (FPP), which is not converted by the extant enzyme. In contrast, the consensus sequence generated from the corresponding multiple sequence alignment was found to be inactive toward both substrates. Mutation of a nonconserved position within the aspartate-rich motif of the reconstructed ancestral cyclases was associated with modest effects on activity and relative substrate specificity (i.e., kcat /KM for GGPP over kcat /KM for FPP). Kinetic analyses performed at different temperatures reveal a loss of substrate saturation, when going from the ancestor with highest thermostability to the modern enzyme. The kinetics data also illustrate how an increase in temperature optimum of biocatalysis is reflected in altered entropy and enthalpy of activation. Our findings further highlight the potential and limitations of applying ASR to biosynthetic machineries in secondary metabolism.

Physiological and yield responses of purple coneflower (Echinacea purpurea (L.) Moench) to nitrogen sources at different levels of irrigation.

Echinacea purpurea (L.) is one of the most important medicinal plants in the world showing different biochemical reactions as affected by drought stress and nitrogen fertilizer. The purpose of this study was to determine the effect of nitrogen on soluble protein, enzyme activities, carotenoids metabolism, greenness and biological yield of the Echinacea purpurea under different levels of irrigation. The experiment was conducted in a research field in Iran during 2013-2015. Irrigation treatments included irrigation after 25, 50 and 75% soil water depletion, and nitrogen sources were no nitrogen (N), nitroxin, 40kgNha-1, 40kgNha-1 + nitroxin and 80kgNha-1. Nitroxin is a biofertilizer including Azotobacter and Azospirillum. The activity of catalase (11.9-21.3mmolg-1fwmin-1), peroxidase (40.1-48.9µmolg-1fwmin-1), polyphenoloxidase (0.79-3.63mmolg-1fwmin-1) increased in both years under drought stress conditions. The lowest greenness (50.1-45.9) was achieved from no-application of nitrogen in the irrigation treatment after 75% water depletion. The results demonstrated the beneficial effects of nitrogen sources on physiological reactions, especially peroxidase, polyphenol oxidase and carotenoids metabolism. It is concluded that irrigation of E. purpurea based on 50% water depletion and 80kg nitrogen as well as the combination of nitroxin and 40kg nitrogen treatments should be an appropriate choice for 2years.

GroEL/ES mediated the in vivo recovery of TRAIL inclusion bodies in Escherichia coli

Inclusion body (IB) formation generates substantial bio-waste in the pharmaceutical industry and remains a major challenge for heterologous protein expression. Although chaperones can be co-expressed to improve soluble protein yield, their contribution to IB processing in vivo has not been thoroughly studied. Here, a GroEL-GroES co-expressing strain and a deficient strain were constructed to study the in vivo recovery of recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The interaction between GroEL/ES and TRAIL was simulated by molecular docking and identified by co-immunoprecipitation. The in vitro cytotoxicity of TRAIL IBs before and after in vivo recovery was subsequently determined by MTT assay. Additionally, IB structures were measured by Fourier transform infrared (FT-IR) spectroscopy and fluorescence spectroscopy. The results showed that after in vivo refolding, IBs retained lower levels of anti-tumor activity and fewer native-like β-sheet structures. Fewer recoverable polypeptides were trapped in IBs after GroEL/ES co-expression and refolding in vivo. Therefore, GroEL/ES mediated the in vivo recovery of TRAIL IBs in Escherichia coli. These results may identify potential uses for IBs and provide additional insight into the detailed mechanisms of in vivo protein recovery.

The Goldilocks Approach: A Review of Employing Design of Experiments in Prokaryotic Recombinant Protein Production.

The production of high yields of soluble recombinant protein is one of the main objectives of protein biotechnology. Several factors, such as expression system, vector, host, media composition and induction conditions can influence recombinant protein yield. Identifying the most important factors for optimum protein expression may involve significant investment of time and considerable cost. To address this problem, statistical models such as Design of Experiments (DoE) have been used to optimise recombinant protein production. This review examines the application of DoE in the production of recombinant proteins in prokaryotic expression systems with specific emphasis on media composition and culture conditions. The review examines the most commonly used DoE screening and optimisation designs. It provides examples of DoE applied to optimisation of media and culture conditions.

Creation of culture media for efficient duckweeds micropropagation (Wolffia arrhiza and Lemna minor) using artificial mathematical optimization models

Recently, computer technologies have provided the researchers with the new approaches for modeling and better understanding the role of the factors that are involved in plant growth in vitro. To develop new models for the optimization of growth conditions, it is reasonable to use plants with a high speed of vegetative in vitro reproduction, such as duckweed (Lemnaceae family). This article focuses on the trophic levels of the two types of duckweeds (Wolffia arrhiza and Lemna minor). Using the development of the optimal modeling of the biological processes we have obtained the prescriptions for individually-balanced culture medium that enable 3.0 higher yields of the total soluble protein from each of the populations for both types of Lemnaceae.

Rapid method for an enhanced recovery of biologically active human phospholipid scramblase1 from inclusion bodies

Human phospholipid scramblase 1 (hPLSCR1) is an ATP independent, Ca2+ dependent transmembrane protein mediating bidirectional translocation of phospholipids across the lipid bilayer but the mechanism of scrambling is unknown. Determination of the hPLSCR1 structure would help understand the mechanism and its multi-functional property. Recombinant hPLSCR1 forms inclusion bodies (IBs), when over-expressed in E. coli BL21 (DE3) and recovery of active hPLSCR1 from IBs, were time-consuming and resulted in low protein yield. This study aims to optimize and enhance the expression and purification of active recombinant hPLSCR1 by various strategies. Additives including stabilizers and detergents were added during cell lysis to improve the recovery of soluble hPLSCR1. Five E. coli strains, BL21 (DE3), C43 (DE3), Rosetta, BL21-CodonPlus-RP, and BL21 (DE3) pLysS were screened for maximum yield of soluble protein but localized in IBs. To recover hPLSCR1 from IBs, different additives were added of which, 0.3% N-lauroyl sarcosine (NLS) recovered ∼50% of bioactive hPLSCR1 from IBs. E. coli C43 (DE3) gave higher yields of purified protein (7.76 mg/g cell) followed by E. coli BL21 (DE3) pLysS (5.87 mg/g cell). This report describes a rapid and efficient method for solubilizing membrane proteins from inclusion bodies with a higher recovery.

Efficient osmolyte-based procedure to increase expression level and solubility of infectious hematopoietic necrosis virus (IHNV) nucleoprotein in E. coli.

The nucleoprotein of infectious hematopoietic necrosis virus (IHNV) is considered as the main target antigen for detection of IHNV infection in salmonid fish. This study aimed at improving the expression and solubility of IHNV nucleoprotein (IHNV-NP) in E. coli expression system. The effects of several expression strategies including host strain type, protein expression temperature, heat-shock treatment prior to protein induction, and additives in the growth medium and in the cell lysis buffer were examined. Results showed that bacterial strain type had a great impact on protein expression level, whereas it was not effective in preventing protein aggregation. Production of soluble IHNV-NP was proportionally increased with decreased incubation temperature. Heat-shock treatment prior to protein induction did not change the percent of solubility. For cells grown at low temperature, the presence of additives in the lysis buffer enhanced the solubility of IHNV-NP up to 24%. The highest yield of soluble protein was obtained via incorporation of osmolytes in the growth medium of cells exposed to a mild salt stress, in the following order: sucrose > sorbitol > glycerol > glycine. Soluble protein obtained by the optimized condition was efficiently purified in high yield and successfully detected by two monoclonal antibodies in a sandwich ELISA. Taken together, a combination of proper host strain, low-temperature expression, and timely application of osmolytes in the growth medium provided sufficient quantities of soluble recombinant IHNV-NP that has the potential to be used for diagnostic purposes.

Potential of Extracted Locusta Migratoria Protein Fractions as Value-Added Ingredients.

Although locusts can be sustainably produced and are nutrient rich, the thought of eating them can be hard to swallow for many consumers. This paper aims to investigate the nutritional composition of Locusta migratoria, including the properties of extracted locust protein, contributing to limited literature and product development opportunities for industry. Locusts sourced from Dunedin, New Zealand, contained a high amount of protein (50.79% dry weight) and fat (34.93%), which contained high amounts of omega-3 (15.64%), creating a desirably low omega-3/omega-6 ratio of 0.57. Three protein fractions including; insoluble locust fraction, soluble locust fraction, and a supernatant fraction were recovered following alkali isoelectric precipitation methodology. Initially, proteins were solubilised at pH 10 then precipitated out at the isoelectric point (pH 4). All fractions had significantly higher protein contents compared with the whole locust. The insoluble protein fraction represented 37.76% of the dry weight of protein recovered and was much lighter in colour and greener compared to other fractions. It also had the highest water and oil holding capacity of 5.17 mL/g and 7.31 mL/g, possibly due to larger particle size. The high supernatant yield (56.60%) and low soluble protein yield (9.83%) was unexpected and could be a result of experimental pH conditions chosen.

Expression and Purification of Protein Complexes Suitable for Structural Studies Using Mammalian HEK 293F Cells.

Prokaryotic expression systems have been widely used to express proteins for structural studies. Such expression systems have the advantage of being economical, straightforward and fast. However, for many eukaryotic proteins and particularly protein complexes, bacterial expression systems do not produce significant yields of soluble protein. This may result from failure to efficiently transcribe/translate the required protein or may result from the formation of insoluble aggregates known as inclusion bodies. Mammalian expression systems can often produce natively folded proteins, sometimes with native post-translational modifications. However, such expression systems are underutilized due to the perception that they are costly, technically challenging and result in limited protein yields. In fact, HEK 293F cells are straightforward to grow, transfect with high efficiency and often produce significant yields of recombinant proteins. In this unit, we describe a method to express and purify milligram quantities of a human protein complex from HEK 293F cells grown in suspension transiently transfected with the appropriate plasmids. © 2017 by John Wiley & Sons, Inc.

Prokaryotic expression and action mechanism of antimicrobial LsGRP1C recombinant protein containing a fusion partner of small ubiquitin-like modifier.

Antimicrobial peptides (AMPs) are peptides exhibiting broad-spectrum antimicrobial activities and considered as potential therapeutic agents. LsGRP1C, a novel AMP derived from defense-related LsGRP1 protein of Lilium, was proven to inhibit kinds of bacteria and fungi via alteration of microbial membrane permeability and induction of fungal programmed cell death-like phenomena by in vitro assays using synthetic LsGRP1C. In this study, the prokaryotic production of LsGRP1C recombinant protein containing an N-terminal fusion partner of the yeast small ubiquitin-like modifier (SUMO) was achieved by using optimized Escherichia coli host and purification buffer system, which lead to a high yield of soluble SUMO-LsGRP1C fusion protein. In vitro assay revealed that E. coli-expressed SUMO-LsGRP1C exhibited even better antifungal activity as compared to synthetic LsGRP1C. Meanwhile, the ability of SUMO-LsGRP1C in conducting fungal membrane permeabilization and programmed cell death was verified by SYTOX Green staining and 4',6-diamidino-2-phenylindole staining/terminal deoxynucleotidyl transferase dUTP nick-end labeling assays, respectively, indicating that E. coli-expressed SUMO-LsGRP1C shares identical modes of action with synthetic LsGRP1C. Herein, this E. coli expression system enables the effective and convenient production of antimicrobial LsGRP1C in a form of SUMO-fused recombinant protein.

Combined pretreatment of electrolysis and ultra-sonication towards enhancing solubilization and methane production from mixed microalgae biomass

This study investigated the effect of combination of pretreatment methods such as ultra-sonication and electrolysis for the minimum energy input to recover the maximal carbohydrate and solubilization (in terms of sCOD) from mixed microalgae biomass. The composition of the soluble chemical oxygen demand (COD), protein, carbohydrate revealed that the hydrolysis method had showed positive impact on the increasing quantity and thus enhanced methane yields. As a result, the combination of these 2 pretreatments showed the greatest yield of soluble protein and carbohydrate as 279 and 309mg/L, which is the recovery of nearly 85 and 90% in terms of total content of them. BMP tests showed peak methane production yield of 257mL/gVSadded, for the hydrolysate of combined pretreatment as compared to the control experiment of 138mL/gVS added.

A new kinetic model of ultrasound-assisted pretreatment on rice protein

The aim of this study was to establish a new model to reflect the real ultrasound pretreatment process on rice protein (RP). In this study, ultrasound treatment affected the yield of soluble protein of RP significantly and also improved the enzymatic hydrolysis efficiently. However, the aggregation of soluble protein occurred when ultrasonic power density and temperature reached a high level. The traditional model cannot reflect the real pretreatment process on RP due to the aggregation of protein. In this paper, a modified kinetic model was thus established by introducing a parameter of aggregation coefficient based on the Fick’s second law, suggesting the two coefficients were highly dependent on the ultrasonic power density and temperature. Besides, the results of scanning electron microscope (SEM) both the untreated and samples pretreated by ultrasound indicated that the enzymatic hydrolysis efficiency was influenced by changing the structure of RP. Thereto, ultrasound treatment at proper conditions can shorten the enzymolysis time of RP and improve the efficiency of hydrolysis.

Effect of Pgrs and Nutrients on Growth, Nitrate Reductase Activity, Soluble Protein, Proline Content and Yield in Bhendi Hybrid Under Salinity

An experiment was conducted to study the effect of PGRs { gibberellic acid (10 ppm), brassinolide (0.5 ppm), salicylic acid (100 ppm), ascorbic acid (100 ppm), benzyl amino purine (5 ppm)} and nutrients { K2SO4 (0.5%) + FeSO4 (0.5%) + Borax (0.3%) mixture and 19:19:19 (1%) mixture} on growth, NR enzyme activity, proline, soluble protein content and yield of bhendi hybrid (COBh H 1) under saline condition. The treatments were given at 25 and 45 DAS as foliar spray. The results showed that, nitrate reductase activity, soluble protein and plant growth were reduced under saline condition where as proline content was increased compared to absolute control. Foliar application of PGRs and nutrients enhanced the NR activity, proline, soluble protein and plant growth under saline condition. Among the PGRs and nutrients, foliar application of salicylic acid (100 ppm) and brassinolide (0.5 ppm) showed the better performance to mitigate the effect of salinity.

Expression, purification and functionality of bioactive recombinant human vascular endothelial growth factor VEGF165 in E. coli

Vascular endothelial growth factor (VEGF) is associated with tumour growth and metastasis. Because VEGF is the major player in both angiogenesis and vascular permeability and the most explored factor in angio-inhibitory therapies, many expression procedures have been developed to produce functional VEGF165 in convenient yield. In this study, recombinant human VEGF165 was cloned and expressed in Escherichia coli (BL21)-DE3 cells and large scale production was performed by fermentation. A high yield of active soluble protein was obtained after protein extraction employing both lysozyme and sonication treatment. Inclusion bodies were also isolated from the cell lysate and subjected to a simple protocol of solubilisation and refolding. Single-step purification was performed using nickel affinity chromatography and the purified proteins were able to recognize monoclonal Anti-poly-His antibody. The biological activity of the VEGF165 was successfully tested using the Chicken chorioallantoic membrane assay, wound-healing migration and proliferation assay on human umbilical vein endothelial cells (HUVEC).

Biorefinery of microalgal soluble proteins by sequential processing and membrane filtration

A mild biorefinery process was investigated on the microalga Nannochloropsis gaditana, to obtain an enriched fraction of water soluble proteins free from chlorophyll. After harvesting, a 100g.L−1 solution of cells was first subjected to cell disruption by either high-pressure homogenization (HPH) or enzymatic treatment (ENZ). HPH resulted in a larger release of proteins (49%) in the aqueous phase compared to the Alcalase incubation (35%). In both cases, an ultrafiltration/diafiltration (UF/DF) was then performed on the supernatant obtained from cell disruption by testing different membrane cut-off (1000kDa, 500kDa and 300kDa). After optimising the process conditions, the combination of ENZ→UF/DF ended in a larger overall yield of water soluble proteins (24.8%) in the permeate compared to the combination of HPH→UF/DF (17.4%). A gel polarization model was implemented to assess the maximum achievable concentration factor during ultrafiltration and the mass transfer coefficient related to the theoretical permeation flux rate.

Development of a vaccine against Streptococcus agalactiae in fish based on truncated cell wall surface anchor proteins.

Streptococcus agalactiae is an important fish pathogen and a leading cause of major economic losses to the aquaculture industry worldwide. In the present study, the two truncated recombinant proteins of cell wall surface anchor family of S agalactiae, CWSAP465 and CWSAP1035, were expressed in Escherichia coli, and their immunogenicity and efficacy against the bacterium were evaluated in tilapia and turbot. The results showed that the prokaryotic expression of the two constructs, p32a-CWSAP465 and p32a-CWSAP1035, gave rise to a high yield of soluble proteins with good immunogenicity. The immunisation-challenge study revealed that tilapia and turbot immunised with recombinant truncated proteins produced high levels of antibodies with a peak at four weeks after immunisation and were protected from a challenge by a virulent S agalactiae at a dose of 1×109 colony forming units/ml. The recombinant truncated proteins had higher efficacy than the whole-cell inactivated vaccine. Therefore, the study demonstrated that CWSAP465 and CWSAP1035 are two viable vaccine candidates against S agalactiae in fish.