Production Of Soluble Protein Research Articles

Enhanced recombinant Sulfurihydrogenibium yellowstonense carbonic anhydrase activity and thermostability by chaperone GroELS for carbon dioxide biomineralization

Biological carbon fixation is a feasible strategy to reduce atmospheric carbon dioxide levels (CO2). In this platform, carbonic anhydrase (CA) enzyme is employed to accelerate the sequestration of CO2. The present work explored the effect of chaperone GroELS and TrxA-tag on improving soluble expression of the recombinant Sulfurihydrogenibium yellowstonense CA which activity and biomineralization capability were taken into consideration. At first, the expression of GroELS using the inducible T7 promoter and constitutive J23100 promoter were investigated. The results indicated that 1.4 folds increment of soluble protein and 100% of CA activity enhancement were achieved with GroELS co-expression driven by J23100 promoter. Furthermore, the involvement of TrxA fusion tag displayed a significant enhancement of soluble protein production which was about 2.67 times higher than that of original SyCA. Besides, co-expression with GroELS intensified the thermostability of SyCA at 60 °C owing to changes in the structural conformation of the protein, which was proved by an in vitro assay. The SyCA was further entrapped and immobilized into polyacrylamide gel (i.e., PAGE-SyCA). The biomineralization capability of the PAGE-SyCA and whole-cell (WC) was compared in a two-column system. After 5 cycles of reuse, PAGE-SyCA maintained 29.8% activity and formed 774 mg of CaCO3 solids in the B::JG strain. This study presents the recombinant engineering strategies to improve SyCA productivity, activity, thermostability, and effective carbon dioxide conversion.

Alleviation of allelochemical stress-induced growth inhibition and oxidative damage in lettuce under closed hydroponics through electro-degradation

Successive lettuce cultivation in closed hydroponics using the same nutrient solution causes the excess production and accumulation of allelochemicals. The accumulated allelochemicals induce oxidative damage and lipid peroxidation in plants leading to growth inhibition. In this study, we investigated the allelochemicals that induced oxidative damage and lipid peroxidation in lettuce grown in a once used non-renewed nutrient solution (1NR) and a twice used non-renewed nutrient solution (2NR) obtained from the successive cultivation and the alleviation of these damages through electro-degradation (ED). The 1NR solution was used for six weeks for a one-time lettuce cultivation while the 2NR solution was used for twelve weeks for a two-times lettuce cultivation. The results showed that the allelochemical stress caused growth inhibition in the lettuce in both the 1NR and 2NR solutions. It was observed that there was a higher generation of H2O2 and O2.– as well as a lower activity of the antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), and ascorbate peroxidase (APX) in the roots of the plants grown in both the 1NR and 2NR solutions compared to plants grown in the new nutrient solution. The higher level of lipid peroxidation due to the higher MDA (malondialdehyde) content and higher soluble protein content were also observed in the roots of those plants. It was evident that lettuce root damage occurred due to accumulation of the allelochemicals in the 1NR and 2NR solutions. These damaged roots could not function normally nor uptake water and minerals from the culture solution. As a result, retarded lettuce growth was observed in the 1NR and 2NR solutions. The oxidative damage, soluble protein content, lipid peroxidation and ultimately growth retardation were more pronounced in the plants grown in the 2NR solution compared to the plants grown in the 1NR solution. The application of ED to the 1NR and 2NR solutions maintained the plant growth through less oxidative damage, soluble protein production and lipid peroxidation as was observed in the plants grown with the new nutrient solution. Therefore, the ED of a non-renewed culture solution would alleviate the allelochemical stress in lettuce under recycled hydroponics.

Evaluating Protein Engineering Thermostability Prediction Tools Using an Independently Generated Dataset.

Engineering proteins to enhance thermal stability is a widely utilized approach for creating industrially relevant biocatalysts. The development of new experimental datasets and computational tools to guide these engineering efforts remains an active area of research. Thus, to complement the previously reported measures of T50 and kinetic constants, we are reporting an expansion of our previously published dataset of mutants for β-glucosidase to include both measures of TM and ΔΔG. For a set of 51 mutants, we found that T50 and TM are moderately correlated, with a Pearson correlation coefficient and Spearman’s rank coefficient of 0.58 and 0.47, respectively, indicating that the two methods capture different physical features. The performance of predicted stability using nine computational tools was also evaluated on the dataset of 51 mutants, none of which are found to be strong predictors of the observed changes in T50, TM, or ΔΔG. Furthermore, the ability of the nine algorithms to predict the production of isolatable soluble protein was examined, which revealed that Rosetta ΔΔG, FoldX, DeepDDG, PoPMuSiC, and SDM were capable of predicting if a mutant could be produced and isolated as a soluble protein. These results further highlight the need for new algorithms for predicting modest, yet important, changes in thermal stability as well as a new utility for current algorithms for prescreening designs for the production of mutants that maintain fold and soluble production properties.

Multiparameter Analysis Identifies Heterogeneity in Knee Osteoarthritis Synovial Responses.

Synovial membrane inflammation is common in osteoarthritis (OA) and increases cartilage injury. However, synovial fluid and histology studies suggest that OA inflammatory responses are not homogeneous. Greater understanding of these responses may provide new insights into OA disease mechanisms. We undertook this study to develop a novel multiparameter approach to phenotype synovial responses in knee OA. Cell composition and soluble protein production were measured by flow cytometry and multiplex enzyme-linked immunosorbent assay in synovium collected from OA patients undergoing knee replacement surgery (n = 35). Testing disaggregation conditions showed that aggressive digestion improved synovial cell yield and mesenchymal staining by flow cytometry, but it negatively impacted CD4+ T cell and CD56+ natural killer cell staining. Less aggressive digestion preserved these markers and showed highly variable T cell infiltration (range 0-43%; n = 32). Correlation analysis identified mesenchymal subpopulations associated with different nonmesenchymal populations, including macrophages and T cells (CD45+CD11b+HLA-DR+ myeloid cells with PDPN+CD73+CD90-CD34- mesenchymal cells [r = 0.65, P < 0.0001]; and CD45+CD3+ T cells with PDPN+CD73+CD90+CD34+ mesenchymal cells [r = 0.50, P = 0.003]). Interleukin-6 (IL-6) measured by flow cytometry strongly correlated with IL-6 released by ex vivo culture of synovial tissue (r = 0.59, P = 0.0012) and was highest in mesenchymal cells coexpressing CD90 and CD34. IL-6, IL-8, complement factor D, and IL-10 release correlated positively with tissue cellularity (P = 0.0042, P = 0.018, P = 0.0012, and P = 0.038, respectively). Additionally, increased CD8+ T cell numbers correlated with retinol binding protein 4 (P = 0.033). Finally, combining flow cytometry and multiplex data identified patient clusters with different types of inflammatory responses. We used a novel approach to analyze OA synovium, identifying patient-specific inflammatory clusters. Our findings indicate that phenotyping synovial inflammation may provide new insights into OA patient heterogeneity and biomarker development.

Stepwise optimization of recombinant protein production in Escherichia coli utilizing computational and experimental approaches.

Over the past few decades, Escherichia coli (E. coli) remains the most favorable host among the microbial cell factories for the production of soluble recombinant proteins. Recombinant protein production (RPP) via E. coli is optimized at the level of gene expression (expression level) and the process condition of fermentation (process level). Presently, the reported studies do not give a clear view on the selection of methods employed in the optimization of RPP. Here, we have reviewed various optimization methods and their preferences with respect to the factors at expression and process levels to achieve theoptimal levels of soluble RPP. With a greater understanding of these optimization methods, we proposed a stepwise methodology linking the factors from both levels for optimizing the production of soluble recombinant protein in E. coli. The proposed methodology is further explained through five sets of examples demonstrating the optimization of RPP at both expression and process levels.Key Points• Stepwise methodology of optimizing recombinant protein production is proposed.• In silico tools can facilitate the optimization of gene- and protein-based factors.• Optimization of gene- and protein-based factors aids host-vector selection.• Statistical optimization is preferred for achieving optimal levels of process factors.

Bioconversion of chicken feather wastes by keratinolytic bacteria

Rhodococcus erythropolis, Geobacillus stearothermophilus and two Bacillus species (Bacillus pumilis, and Bacillus licheniformis) were evaluated for protease production using feathers as the sole carbon and nitrogen source. The B. licheniformis produced enzymes optimally at 40°C and pH 10.0, while B. pumilis performed optimally at 37°C and pH 7.0. Furthermore, the optimum conditions for enzyme production by G. stearothermophilus were 55°C and pH 7.0–8.0, while for R. erythropolis they were 37°C and pH 7.0. The maximum proteolytic activities of the protease produced by B. licheniformis, G. stearothermophylus and B. pumilis were 50.41, 9.91 and 35.41 U/ml after 48, 72 and 48h of culture, respectively. With R. erythropolis, the maximum enzyme activity was 33.39 U/ml after 96h of culture. Also, the production of soluble protein showed the same pattern as that of proteolytic protease. When the initial pH value was 10.0, culturing the feathers (30g l−1) for four days at 40°C resulted in the maximum production of soluble proteins (8.28mg/ml) by B. licheniformis. These results suggest that new strategies for waste management have emerged after the introduction of keratinases in sustainable material development, which has industrials applications in green technologies.

Longitudinal RNA-Seq Analysis of the Repeatability of Gene Expression and Splicing in Human Platelets Identifies a Platelet SELP Splice QTL.

Longitudinal studies are required to distinguish within versus between-individual variation and repeatability of gene expression. They are uniquely positioned to decipher genetic signal from environmental noise, with potential application to gene variant and expression studies. However, longitudinal analyses of gene expression in healthy individuals-especially with regards to alternative splicing-are lacking for most primary cell types, including platelets. To assess repeatability of gene expression and splicing in platelets and use repeatability to identify novel platelet expression quantitative trait loci (QTLs) and splice QTLs. We sequenced the transcriptome of platelets isolated repeatedly up to 4 years from healthy individuals. We examined within and between individual variation and repeatability of platelet RNA expression and exon skipping, a readily measured alternative splicing event. We find that platelet gene expression is generally stable between and within-individuals over time-with the exception of a subset of genes enriched for the inflammation gene ontology. We show an enrichment among repeatable genes for associations with heritable traits, including known and novel platelet expression QTLs. Several exon skipping events were also highly repeatable, suggesting heritable patterns of splicing in platelets. One of the most repeatable was exon 14 skipping of SELP. Accordingly, we identify rs6128 as a platelet splice QTL and define an rs6128-dependent association between SELP exon 14 skipping and race. In vitro experiments demonstrate that this single nucleotide variant directly affects exon 14 skipping and changes the ratio of transmembrane versus soluble P-selectin protein production. We conclude that the platelet transcriptome is generally stable over 4 years. We demonstrate the use of repeatability of gene expression and splicing to identify novel platelet expression QTLs and splice QTLs. rs6128 is a platelet splice QTL that alters SELP exon 14 skipping and soluble versus transmembrane P-selectin protein production.

Heterologous Biosynthesis of Type II Polyketide Products Using E. coli

The heterologous biosynthesis of complex natural products has enabled access to polyketide, nonribosomal peptide, isoprenoid, and other compounds with wide-spanning societal value. Though several surrogate host systems exist, Escherichia coli is often a preferred choice due to its rapid growth kinetics and extensive molecular biology protocols. However, a persistent challenge to the utilization of E. coli has been the successful in vivo reconstitution of type II polyketide synthase (PKS) systems. In particular, gene expression of the ketosynthase (KS) components of the minimal PKS has consistently yielded insoluble protein products. In the following report, two type II PKS systems were functionally reconstituted in E. coli. The approach to do so relied upon the utilization of the native transcriptional coupling between the dimeric KS subunits, leading to soluble recombinant protein products and successful polyketide biosynthesis. Resulting strains produced 10 mg/L TW95c and 25 mg/L dehydrorabelomycin. Hence, the strategy offers a new option in the biosynthetic engineering efforts for the heterologous production of type II polyketide products using E. coli.

Plant Phenolics and Lectins as Vaccine Adjuvants.

The immune system is responsible for providing protection to the body against foreign substances. The immune system divides into two types of immune responses to study its mechanisms of protection: 1) Innate and 2) Adaptive. The innate immune response represents the first protective barrier of the organism that also works as a regulator of the adaptive immune response, if evaded the mechanisms of the innate immune response by the foreign substance the adaptive immune response takes action with the consequent antigen neutralization or elimination. The adaptive immune response objective is developing a specific humoral response that consists in the production of soluble proteins known as antibodies capable of specifically recognizing the foreign agent; such protective mechanism is induced artificially through an immunization or vaccination. Unfortunately, the immunogenicity of the antigens is an intrinsic characteristic of the same antigen dependent on several factors. Vaccine adjuvants are chemical substances of very varied structure that seek to improve the immunogenicity of antigens. The main four types of adjuvants under investigation are the following: 1) Oil emulsions with an antigen in solution, 2) Pattern recognition receptors activating molecules, 3) Inflammatory stimulatory molecules or activators of the inflammasome complex, and 4) Cytokines. However, this paper addresses the biological plausibility of two phytochemical compounds as vaccine adjuvants: 5) Lectins, and 6) Plant phenolics whose characteristics, mechanisms of action and disadvantages are addressed. Finally, the immunological usefulness of these molecules is discussed through immunological data to estimate effects of plant phenolics and lectins as vaccine adjuvants, and current studies that have implanted these molecules as vaccine adjuvants, demonstrating the results of this immunization.

A Novel Nanobody Scaffold Optimized for Bacterial Expression and Suitable for the Construction of Ribosome Display Libraries.

Single-domain antigen-binding fragments of camelid antibodies, known as VHHs or nanobodies, are widely used affinity reagents. However, their production involving animal immunization is time- and resource-intensive. Starting from a sequence dataset of llama VHHs, we designed a novel scaffold, based on conserved framework sequences, suitable for bacterial nanobody expression and synthetic library construction. The consensus scaffold was validated by grafting the CDRs from two known nanobodies. While maintaining their binding properties, the two chimeric nanobodies showed higher levels of expression and solubility in E. coli when compared to the corresponding wild types. A proof-of-concept synthetic combinatorial library, suitable for ribosome display (RD) selection, was obtained by encoding three randomized complementarity determining regions within the consensus framework. The library, made of linear DNA fragments, has an estimated complexity of > 1012 that is three orders of magnitude higher than common phage display libraries. The bacterial expression of several library clones showed a high production of soluble recombinant proteins. The high complexity of the library, confirmed by sequencing of a subset of clones, as well as a preliminary RD selection of a maltose binding protein binder, indicated this approach as a starting point in the construction of synthetic combinatorial libraries to be used as animal-free tools for the low-cost selection of target-specific nanobodies.

Conversion of the molecular chaperone Spy into a novel fusion tag to enhance recombinant protein expression

The soluble expression of recombinant proteins in Escherichia coli is vital for protein applications in biotechnology and pharmaceuticals. However, the use of E. coli for efficient heterologous protein expression is hampered by several factors, such as poor expression and protein aggregation. Changing the culture or purification conditions may alleviate these issues, but methods based on gene fusion technology offer unique opportunities to improve the production and purification of soluble proteins. Here, we develop a novel fusion tag based on Spy, a newly identified molecular chaperone that functions in the periplasm of E. coli in an ATP-independent manner to prevent protein aggregation and assist in protein folding. We found that the tandem fusion of Spy stands among the well-described best fusion partners, such as MBP and SUMO, in increasing the soluble steady-state levels of six heterologous passenger proteins. Moreover, an easily aggregated passenger protein remained soluble after the removal of the Spy tag, implying that chaperone-dependent folding occurred when the passenger protein was fused to Spy. Our work expands the toolkit of fusion tags and allows them to aid in the production of unstable proteins with industrial or clinical values.

Enhanced hydrolysis-acidification of high-solids and low-organic-content sludge by biological thermal-alkaline synergism

In this study, a biological thermal-alkaline synergistic system was successfully established to enhance the hydrolysis-acidification efficiency of high-solids and low-organic-content sludge (HS-LOC-S). The results indicated that the highest hydrolysis rate was obtained at pH of 12 (52.62%) leading to the highest production of soluble chemical oxygen demand (SCOD) and soluble protein (SP). The highest acidification rate was observed at pH of 10 (32.15%), leading to the highest production of volatile fatty acids (VFAs). At pH of 10, average sludge size reduced by 24.60%, and the proportion of biodegradable dissolved organic matter (DOM) produced by synergistic system increased by 15.82%, when compared with those of raw sludge. Moreover, results of 16S rRNA clearly validated that the relative abundance of hydrolytic and acidogenic microbes (e.g. Tepidimicrobium, Coprothermobacter) abundantly enriched at pH of 10 (49.88%) was greatly higher than others, which was the main reason for its maximum VFAs accumulation.

Selective biodegradation of recalcitrant black chicken feathers by a newly isolated thermotolerant bacterium Pseudochrobactrum sp. IY-BUK1 for enhanced production of keratinase and protein-rich hydrolysates.

Black chicken feathers generated in large amount from poultry and slaughter houses are highly recalcitrant to microbial degradation due to their tough structural nature. A novel keratinolytic bacterium that possessed high affinity for black feather was isolated from chicken manure and identified as Pseudochrobactrum sp. IY-BUK1. Keratinase and feather soluble protein were effectively produced by the free living cells of the bacterium in media containing only black feathers and a mixture of equal amount of black-, brown- and white-coloured feathers. Complete degradation of 5g/L of black feathers was completed in 3days following optimisation of physico-chemical conditions. However, the bacterium selectively completed the degradation of black feather in a medium containing mixture of feathers in 144h leaving behind approximately 33% and 45% of brown and white feathers in the medium respectively. Gellan gum-immobilised cells of strain IY-BUK1 enhanced the keratinase production by about 150% and were used repeatedly for ten cycles to degrade 5g/L of black feather in a semi continuous fermentation of 18h per cycle with enhanced and stable production of soluble protein. The study demonstrated the potential use of Pseudochrobactrum sp. IY-BUK1 not only in biodegradation of highly recalcitrant black feathers, but also in producing keratinase enzymes and valuable soluble proteins for possible industrial usage.

Drought Tolerance of Soybean (Glycine max L. Merr.) by Improved Photosynthetic Characteristics and an Efficient Antioxidant Enzyme Activities Under a Split-Root System.

Water deficiency significantly affects photosynthetic characteristics. However, there is little information about variations in antioxidant enzyme activities and photosynthetic characteristics of soybean under imbalanced water deficit conditions (WDC). We therefore investigated the changes in photosynthetic and chlorophyll fluorescence characteristics, total soluble protein, Rubisco activity (RA), and enzymatic activities of two soybean varieties subjected to four different types of imbalanced WDC under a split-root system. The results indicated that the response of both cultivars was significant for all the measured parameters and the degree of response differed between cultivars under imbalanced WDC. The maximum values of enzymatic activities (SOD, CAT, GR, APX, and POD), chlorophyll fluorescence (Fv/Fm, qP, ɸPSII, and ETR), proline, RA, and total soluble protein were obtained with a drought-tolerant cultivar (ND-12). Among imbalanced WDC, the enhanced net photosynthesis, transpiration, and stomatal conductance rates in T2 allowed the production of higher total soluble protein after 5 days of stress, which compensated for the negative effects of imbalanced WDC. Treatment T4 exhibited greater potential for proline accumulation than treatment T1 at 0, 1, 3, and 5 days after treatment, thus showing the severity of the water stress conditions. In addition, the chlorophyll fluorescence values of FvFm, ɸPSII, qP, and ETR decreased as the imbalanced WDC increased, with lower values noted under treatment T4. Soybean plants grown in imbalanced WDC (T2, T3, and T4) exhibited signs of oxidative stress such as decreased chlorophyll content. Nevertheless, soybean plants developed their antioxidative defense-mechanisms, including the accelerated activities of these enzymes. Comparatively, the leaves of soybean plants in T2 displayed lower antioxidative enzymes activities than the leaves of T4 plants showing that soybean plants experienced less WDC in T2 compared to in T4. We therefore suggest that appropriate soybean cultivars and T2 treatments could mitigate abiotic stresses under imbalanced WDC, especially in intercropping.

An improved method for the heterologous production of soluble human ribosomal proteins in Escherichia coli

Human ribosomal proteins play important structural and functional roles in the ribosome and in protein synthesis. An efficient method to recombinantly produce and purify these proteins would enable their full characterisation. However, the production of human ribosomal proteins can be challenging. The only published method about the recombinant production of human ribosomal proteins involved the recovery of proteins from inclusion bodies, a process that is tedious and may lead to significant loss of yield. Herein, we explored the use of different Escherichia coli competent cells and fusion protein tags for the recombinant production of human ribosomal proteins. We found that, by using thioredoxin as a fusion protein, soluble ribosomal protein could be obtained directly from cell lysates, thus leading to an improved method to recombinantly produce these proteins.

Relationship between Protein Expression Pattern and Host Metabolome Perturbation as Monitored by Two‐Dimensional NMR Spectroscopy

In producing large amounts of heterologous proteins, researchers most often use Escherichia coli as a host thanks to its extensively studied genetics, simple growth procedure, and low cost. However, the desired protein is often produced only in a form of inclusion bodies. Researchers have tried to devise a way to circumvent such a problem, and the ones using fusion partners seem to be the most successful. Based on our previous observation that the host metabolome was related to the outcomes of protein expression patterns, we proceeded to perturb the metabolome by applying a salt stress to see if we could shake up metabolite compositions to make them better suited for soluble expression of the target protein. We examined a subset of the metabolites which had been partially labeled with input 13C‐glucose. We tested 11 genes under 4 different NaCl concentrations, and identified 18 metabolites using the heteronuclear single quantum coherence NMR experiment. Most of the proteins kept their expression profiles unchanged, but two proteins were converted from inclusion bodies to a soluble form with increased NaCl concentration. Through the statistical analysis, we could identify a region where the soluble protein production was favored in the metabolite space. We hope that this work would provide an alternative strategy to produce the recombinant proteins in their soluble or native forms, not only in E. coli but also in other hosts.

Evaluation of scFv protein recovery from E. coli by in vitro refolding and mild solubilization process

BackgroundThe production of therapeutically active single chain variable fragment (scFv) antibody is still challenging in E. coli due to the aggregation propensity of recombinant protein into inclusion bodies (IBs). However, recent advancement of biotechnology has shown substantial recovery of bioactive protein from such insoluble IBs by solubilization and refolding processes. In addition, gene fusion technology has also widely been used to improve the soluble protein production using E. coli. This study demonstrates that mild-solubilization and in vitro refolding strategies, both are capable to recover soluble scFv protein from bacterial IBs, although the degree of success is greatly influenced by different fusion tags with the target protein.ResultsIt was observed that the most commonly used fusion tag, i.e., maltose binding protein (MBP) was not only influenced the cytoplasmic expression in E. coli but also greatly improved the in vitro refolding yield of scFv protein. On the other hand, mild solubilization process potentially could recover soluble and functional scFv protein from non-classical IBs without assistance of any fusion tag and in vitro refolding step. The recovery yield achieved by mild solubilization process was also found higher than denaturation–refolding method except while scFv was refolded in fusion with MBP tag. Concomitantly, it was also observed that the soluble protein achieved by mild solubilizationprocess was better structured and functionally more active than the one achieved by in vitro refolding method in the absence of MBP tag or refolding enhancer.ConclusionsMaltose binding protein tagged scFv has shown better refolding and solubility yields as compare to mild solubilization process. However, in terms of cost, time and tag free nature, mild solubilization method for scFv recovery from bacterial IBs is considerable for therapeutic application and further structural studies.

Preparation and Evaluation of activity of Highly-soluble Anticancer Protein Drug TATm-survivin (T34A)

TATm-survivin (T34A), an anticancer protein drug, displayed pro-apoptotic bioactivity against various cancer cells. It was expressed in Escherichia coli as inclusion bodies. To test the bioactivity of soluble TATm-survivin (T34A), a feasible strategy was first developed for the soluble expression and purification. Effect of zinc ion and induction temperature was investigated to improve the solubility and overall production level of TATm-survivin (T34A). High solubility (92 %) was achieved by addition of zinc ion and temperature downshift after induction. An efficient protocol of purification was established by heat release, ammonium sulphate precipitation, anion exchange and heparin affinity chromatography. Purified TATm-survivin (T34A) was obtained with a purity of 96 %, which inhibited the proliferation of human pancreas carcinoma cell lines SW1990. In comparison to denatured TATm-survivin (T34A), soluble TATm-survivin (T34A) did not exhibit higher bioactivity as expected. This study represented a novel strategy to obtain highly soluble form of TATm-survivin (T34A) and would provide useful information for production of other soluble tat-mediated fusion proteins.

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.

Production of Stabilized Antibody Fragments in the E. coli Bacterial Cytoplasm and in Transiently Transfected Mammalian Cells.

Monoclonal antibodies (mAbs) are currently the fastest growing class of therapeutic proteins. Parallel to full-length IgG format the development of recombinant technologies provided the production of smaller recombinant antibody variants. The single-chain variable fragment (scFv) antibody is a minimal form of functional antibody comprised of the variable domains of immunoglobulin light and heavy chains connected by a flexible linker. In most cases, scFvs are expressed in the periplasm bacterium E. coli. The production of soluble scFvs is more effective in quantity, however, under the reducing conditions of the E. coli bacterial cytoplasm it is inefficient because of the inability of the disulfide bonds to form. Hence, scFvs are either secreted to the periplasm as soluble proteins or expressed in the cytoplasm as insoluble inclusion bodies and recovered by refolding. The cytoplasmic expression of scFvs as a C-terminal fusion to maltose-binding protein (MBP) provided the high-level production of stable, soluble, and functional fusion protein. The below protocol provides the detailed description of MBP-scFv production in E. coli utilizing two expression systems: pMALc-TNN and pMALc-NHNN. Although the MBP tag does not disrupt the most of antibody activities, the MBP-TNN-scFv product can be cleaved by Tobacco Etch Virus (TEV) protease in order to obtain untagged scFv.The second protocol is for efficient production of Fab antibody fragments as MBP fusion proteins secreted by transiently transfected mammalian cells. While transient transfection is a fast and effective way of obtaining several mgs of antibody for initial screening and validation of antibodies, some antibody sequences express poorly or not at all. For such antibodies, fusion to MBP provides an effective approach for solving the expression problem.