A novel fed-batch based strategy for enhancing cell-density and recombinant cyprosin B production in bioreactors

Nowadays, the dairy industry is continuously looking for new and more efficient clotting enzymes to create innovative products. Cyprosin B is a plant aspartic protease characterized by clotting activity that was previously cloned in Saccharomyces cerevisiae BJ1991 strain. The production of recombinant cyprosin B by a batch and fed-batch culture was compared using glucose and galactose as carbon sources. The strategy for fed-batch cultivation involved two steps: in the first batch phase, the culture medium presented glucose 1 % (w/v) and galactose 0.5 % (w/v), while in the feed step the culture medium was constituted by 5 % (w/v) galactose with the aim to minimize the GAL7 promoter repression. Based on fed-batch, in comparison to batch growth, an increase in biomass (6.6-fold), protein concentration (59 %) and cyprosin B activity (91 %) was achieved. The recombinant cyprosin B was purified by a single hydrophobic chromatography, presenting a specific activity of 6 × 10⁴ U·mg^?1, corresponding to a purification degree of 12.5-fold and a recovery yield of 16.4 %. The SDS-PAGE analysis showed that recovery procedure is suitable for achieving the purified recombinant cyprosin B. The results show that the recombinant cyprosin B production can be improved based on two distinct steps during the fed-batch, presenting that this strategy, associated with a simplified purification procedure, could be applied to large-scale production, constituting a new and efficient alternative for animal and fungal enzymes widely used in cheese making.     

High-throughput FTIR-based bioprocess analysis of recombinant cyprosin production

To increase the knowledge of the recombinant cyprosin production process in Saccharomyces cerevisiae cultures, it is relevant to implement efficient bioprocess monitoring techniques. The present work focuses on the implementation of a mid-infrared (MIR) spectroscopy-based tool for monitoring the recombinant culture in a rapid, economic, and high-throughput (using a microplate system) mode. Multivariate data analysis on the MIR spectra of culture samples was conducted. Principal component analysis (PCA) enabled capturing the general metabolic status of the yeast cells, as replicated samples appear grouped together in the score plot and groups of culture samples according to the main growth phase can be clearly distinguished. The PCA-loading vectors also revealed spectral regions, and the corresponding chemical functional groups and biomolecules that mostly contributed for the cell biomolecular fingerprint associated with the culture growth phase. These data were corroborated by the analysis of the samples’ second derivative spectra. Partial least square (PLS) regression models built based on the MIR spectra showed high predictive ability for estimating the bioprocess critical variables: biomass (R ² = 0.99, RMSEP 2.8%); cyprosin activity (R ² = 0.98, RMSEP 3.9%); glucose (R ² = 0.93, RMSECV 7.2%); galactose (R ² = 0.97, RMSEP 4.6%); ethanol (R ² = 0.97, RMSEP 5.3%); and acetate (R ² = 0.95, RMSEP 7.0%). In conclusion, high-throughput MIR spectroscopy and multivariate data analysis were effective in identifying the main growth phases and specific cyprosin production phases along the yeast culture as well as in quantifying the critical variables of the process. This knowledge will promote future process optimization and control the recombinant cyprosin bioprocess according to Quality by Design framework.     

Overexpression and characterization of cyprosin B in transformed suspension cells of <Emphasis Type="Italic">Cynara cardunculus</Emphasis>

Cynara cardunculus suspension cells were transformed by particle bombardment to overexpress the cypro11 gene coding for cyprosin B. Green fluorescent protein, used as a visual reporter through mgfp4-ER gene, facilitates the screening of transformed cells at the initial stages when antibiotics cause generalized cell death. mgfp4-ER lacks a cryptic intron and has an endoplasmic reticulum target sequence, these traits conferring an adequate use as screenable marker for transformed cells. Selected transformed cells, grown in a bioreactor, produced 3.8 g dcw l⁻¹ of biomass, 80 mg l⁻¹ of total protein and 2,060 U ml⁻¹ of enzymatic activity. Specific activity of cyprosin B, purified by anionic-exchange chromatography, was 215 U mg⁻¹ with a purification degree of 8.3-fold. The cyprosin B activity is optimal at 42°C for pH 5.1 and is inhibited by pepstatin A. The results encourage the overexpression of cypro11 gene in transformed C. cardunculus cells leading to high yields of cyprosin B production in bioreactor, which can be considered adequate for industrial production.     

Systematic evaluation of single mismatch stability predictors for fluorescence in situ hybridization

The mismatch discrimination potential of probes in fluorescence in situ hybridization can be defined as the difference between the melting formamide points of perfect complementary and mismatched duplexes (Delta[FA](m)). Using a combined experimental and theoretical approach, Delta[FA](m) was determined for a set of 35 mismatched probes targeting seven locations in the 16S rRNA of Escherichia coli. The mismatches were created by changing single nucleotides on the probes, while maintaining the target unmodified. Estimated Delta[FA](m) values were used to systematically evaluate four predictors of mismatch stability: weighted mismatch (WM) scores from the software arb, published statistical summary of microarray hybridizations, free energy of mismatch stability (DeltaDeltaG degrees (1)) and theoretical Delta[FA](m) estimations obtained with a thermodynamic model. Based on the predictors' ability to explain variability in Delta[FA](m) and to discriminate weak mismatches from strong ones, DeltaDeltaG degrees (1) and WM scores from arb (with an updated set of relative strength parameters) were demonstrated to be adequate estimators of mismatch stability, with DeltaDeltaG degrees (1) offering the benefit of capturing the variability associated with nearest-neighbour effects and being compatible with thermodynamic models of in situ hybridization. The use of DeltaDeltaG degrees (1) and WM in probe design was illustrated as a tool that complements experimental design approaches.     

Study of the composting process of winery and distillery wastes using multivariate techniques

Physico-chemical, chemical and biological parameters were studied throughout the composting process of four winery and distillery composts and the data set of compost characteristics was analysed using multivariate techniques: factorial analysis (FA) and linear discriminant analysis (LDA), in order to classify the different parameters studied and thus, to establish those that better describe the composting process of this type of wastes. Through factorial analysis (FA) of the parameters studied throughout the composting process, four components that explained 85.6% of the variability were established. The parameters associated to compost maturity, agronomic character, water-soluble fraction and ammonia and temperature increment were grouped in the components F1, F2, F3 and F4, respectively, which can reduce the number of determinations needed to ascertain the maturity and quality of the composts. In addition, the linear discriminant analysis on the factorial components makes possible to classify the four composts with a percentage of success around 95%.     

肿瘤相关抗原基因HCA520重组蛋白的表达纯化及其肝癌患者血清中相应抗体的分析

HCA5 2 0是用SEREX(serologicalidentificationofrecombinantcDNAexpressingcloning)方法 ,即用肝癌病人血清从肝癌组织cDNA表达文库中筛选得到的肝癌相关性抗原编码基因 .利用RT PCR技术检测了HCA5 2 0mRNA在各种组织中的分布情况 ,构建了GST融合表达载体并且用亲和层析的方法纯化表达的融合蛋白 .最后用Western印迹检测了重组蛋白的免疫反应性 ,用斑点印迹检测了肝癌病人血清中HCA5 2 0的天然抗体的存在情况 .结果表明 ,HCA5 2 0在各种组织中呈丰度差异分布 ,构建好的pGEX 4T 3重组载体经IPTG诱导后高效表达GST HCA5 2 0融合蛋白 ,其分子量约 4 9kD .经GST Agarose亲和层析 ,重组蛋白得到高度纯化 .Western印迹证实 ,纯化蛋白为目的重组蛋白 ,重组蛋白具有与天然蛋白相同或相似的免疫反应性 .斑点印迹分析表明 ,2 0份肝癌病人血清中有 1份HCA5 2 0抗体阳性 ,而 4份正常人均为阴性 .HCA5 2 0的足量提供 ,可用以研究其在致癌中的作用 ,并可以免疫动物制备抗体 .它作为抗原 ,分析其抗体在不同肿瘤病人中的表达情况 ,评估其在临床肿瘤诊断中的作用     

Comparison of the effect of some phenolic compounds on wheat coleoptile section growth with their effect on iaa-oxidase activity

P-coumaric acid (HCA), 2,4-dichlorophenol (DCP) and resorcionol acted as cofactors for IAA-oxidase isolated from young wheat plants. Ferulic acid (FA) and 3,4-dihydroxybenzoic acid (DHBA) induced a lag phase prior to IAA oxidation. HCA, FA (0.2-1 mg ml^-1) and DCP (0.03-1 mg ml^-1) strongly inhibited wheat coleoptile section growth. DHBA (0.01-1 mg ml^-1) slightly stimulated it and resorcinol was without effect. HCA inhibited IAA-induced growth of coleoptile sections and FA stimulated it at low IAA levels and inhibited it at higher ones. DHBA, DCP and resorcinol did not affect IAA-induced growth of coleoptile sections.     

Characterization and expression analysis of the aspartic protease gene family of Cynara cardunculus L

Cardosin A and cardosin B are two aspartic proteases mainly found in the pistils of cardoon Cynara cardunculus L., whose flowers are traditionally used in several Mediterranean countries in the manufacture of ewe's cheese. We have been characterizing cardosins at the biochemical, structural and molecular levels. In this study, we show that the cardoon aspartic proteases are encoded by a multigene family. The genes for cardosin A and cardosin B, as well as those for two new cardoon aspartic proteases, designated cardosin C and cardosin D, were characterized, and their expression in C. cardunculus L. was analyzed by RT-PCR. Together with cardosins, a partial clone of the cyprosin B gene was isolated, revealing that cardosin and cyprosin genes coexist in the genome of the same plant. As a first approach to understanding what dictates the flower-specific pattern of cardosin genes, the respective gene 5' regulatory sequences were fused with the reporter beta-glucuronidase and introduced into Arabidopsis thaliana. A subsequent deletion analysis of the promoter region of the cardosin A gene allowed the identification of a region of approximately 500 bp essential for gene expression in transgenic flowers. Additionally, the relevance of the leader intron of the cardosin A and B genes for gene expression was evaluated. Our data showed that the leader intron is essential for cardosin B gene expression in A. thaliana. In silico analysis revealed the presence of potential regulatory motifs that lay within the aforementioned regions and therefore might be important in the regulation of cardosin expression.     

Identifiability and retrievability of unique parameters describing intrinsic Andrews kinetics

A key factor contributing to the variability in the microbial kinetic parameters reported from batch assays is parameter identifiability, i.e., the ability of the mathematical routine used for parameter estimation to provide unique estimates of the individual parameter values. This work encompassed a three-part evaluation of the parameter identifiability of intrinsic kinetic parameters describing the Andrews growth model that are obtained from batch assays. First, a parameter identifiability analysis was conducted by visually inspecting the sensitivity equations for the Andrews growth model. Second, the practical retrievability of the parameters in the presence of experimental error was evaluated for the parameter estimation routine used. Third, the results of these analyses were tested using an example data set from the literature for a self-inhibitory substrate. The general trends from these analyses were consistent and indicated that it is very difficult, if not impossible, to simultaneously obtain a unique set of estimates of intrinsic kinetic parameters for the Andrews growth model using data from a single batch experiment.     

Expression and purification of thioredoxin (TrxA) and thioredoxin reductase (TrxB) from Brevibacillus choshinensis

Brevibacillus choshinensis (formerly Bacillus brevis) is a protein-hyperproducing bacterium and has been used for commercial protein production. Here, we cloned thioredoxin (trxA) and thioredoxin reductase (trxB) genes from B. choshinensis, and expressed the gene products in Escherichia coli with an amino-terminal hexa-His-tag for purification and characterization. His-TrxA and His-TrxB were purified to homogeneity with one-step Ni-NTA affinity column chromatography, and the two recombinant proteins showed identical specific activity with or without removal of the amino-terminal His-tag, indicating that the extrasequence containing the hexa-His-tag did not affect their enzymatic activities. The E. coli expression system used here resulted in a 40-fold increase in production of His-TrxB protein compared to the level of native TrxB produced in non-recombinant B. choshinensis cells. TrxA and TrxB proteins with carboxy-terminal His-tag (TrxA-His and TrxB-His) were successfully expressed in B. choshinensis and were purified by Ni-NTA column chromatography. Co-expression of TrxA-His with recombinant human epidermal growth factor (hEGF) in B. choshinensis promoted the extracellular production of hEGF by up to about 200%.     

Effects of oxygen and ethanol on recombinant yeast fermentation for hepatitis B virus surface antigen production: modeling and simulation studies

A model was formulated to examine the competitive growth of two phenotypes (Leu(+) and Leu(-)) and the product formation with recombinant Saccharomyces cerevisiae strain DBY-745, which contains the shuttle vector pYGH3-16-s with the foreign gene HBsAg (hepatitis B virus surface antigen) as well as experimental fedbatch fermentation data. The important state variables and the process parameters evaluated include (1) the ratio of the plasmid-free cell concentration to the plasmid-containing cell concentration (rho = X(-)X(+)), (2) the expression of human hepatitis B surface antigen g (CH), (3) the glucose consumption (S), (4) the ethanol production (/), (5) the change of working volume (V) in the fermentor, (6) the different specific growth rates of two phenotype cells, and (7) the plasmid loss frequency coefficient (alpha ). These variables and other parameters were carefully defined, their correlations were studied, and a mathematical model using a set of nonlinear ordinary differential equations (ODEs) for fed-batch fermentation was then obtained based on the theoretical considerations and the experimental results. The extended Kalman filter (EKF) methods was applied for the best estimate of these variables based on the experimentally observable variables: rhoV, and g (CH). Each of these variable was affected by random measuring errors under the different operating conditions. Simulation results presented for verification of the model agreed with our observations and provided useful information relevant to the operation and the control of the fedbatch recombinant yeast fermentation. The method of predicting an optimal profile of the cell growth was also demonstrated under the different dissolved oxygen concentrations. (c) 1993 John Wiley & Sons, Inc.     

A NEW CELL CLONE DERIVED FROM TRICHOPLUSIA NI TN5B1–4 CELLS

Abstract  The characteristics of a cultured cell line do not always remain stable and may change upon continuous passage. Most continuous cell lines, even after cloning, possess several genotypes that are constantly changing. There are numerous selective and adaptive culture processes, in addition to genetic instability, that may improve phenotypic change in cell growth, virus susceptibility, gene expression, and production of virus. Similar detrimental effects of long term passaging of insect cells have also been reported for continuous cell lines, for example, Tn5B1–4 cells, which are the most widely used for the baculovirus expression vector system (BEVS), provide superior production of recombinant proteins, however, this high productivity may be more evident in low passage cells. In this paper, we describe the isolation of a cell clone, Tn5B-40, from low passage Tn5B1–4 cells. The growth characteristics, productions of virus, and high level of recombinant protein productions were determined. The results showed the susceptibility of both clone and Tn5B1–4 cells to wild-type AcNPV was approximately the same rate with over 95% of infection; when the cloned cells were infected with recombinant baculoviruses expressing ß -galactosidase and secreted alkaline phosphatase (SEAP), expression of the recombinant proteins from the cloned cells exceeded that from the parental Tn5B1–4 cells.     

Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study

Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.     

Enhanced production of human Cytochrome P450 2C9 by <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21(DE3)pLysS through the novel use of grey relational analysis and Plackett–Burman design

Cytochrome P450 (CYP, P450) 2C9 is one of three human microsomal CYPs in subfamily 2C that contribute extensively to the hepatic metabolism of therapeutic drugs. The enhancement of recombinant CYP2C9 expression of a transformed E. coli strain is essential for the development of an efficient large-scale bioprocess. The recombinant CYP2C9 production is influenced by various factors, especially the medium components. The aim of this study is to optimize the culture medium of the recombinant E. coli, to determine the influence order of 11 factors, and to improve the yield of heterologously expressed CYP2C9. Plackett–Burman (PB) design, a statistical methodology, was used to screen 11 nutrients and medium components for the production of human CYP2C9 from E. coli BL21(DE3)pLysS harboring plasmid pCW2C9dH in aerobic shaking flask cultures. The experimental data were subject to statistical analysis for calculating the regression coefficients. The matrix of PB design was also used to construct the series that is needed for grey relational analysis (GRA), an approach totally different from traditional statistical analysis used to calculate the relational grade between two sequence data, to determine the influence priority of 11 parameters. Influence priority: glycerol > δ-ALA > IPTG = ampicillin > chloramphenicol = inoculum density > peptone > thiamine > trace elements > NH₄Cl > MgSO₄. The coefficient for the individual factor estimated from PB design agrees the conclusion of GRA. This is the first report that combined two powerful analysis methods for the enhanced production of recombinant protein.     

用遗传算法进行重组大肠杆菌发酵动力学的分析

重组大肠杆菌在诱导表达人表皮生长因子的过程促使细菌的生长受到抑制,一部分重组菌丧失了分裂能力,但仍保持着一定的代谢活力,分离成为存活但不能培养的细菌,根据大肠杆菌在表达外源蛋白过程中细胞生理状态的不同将细菌分为三类,提出一个描述诱导表达过程中重组大肠杆菌分离、生长的动力学模型．应用遗传算法对不同底物浓度的细胞生长、分离和产物合成的动力学参数进行了有效地估计,避免了传统算法可能陷于局部最优的问题,模型计算结果与实验结果吻合良好．分离模型在初始糖浓为5-20g/L的范围内可以较好地描述发酵过程中细胞生长、分离和目标产物表达的过程并具有一定的预测能力．     

Development of biomarkers based on diet-dependent metabolic serotypes: practical issues in development of expert system-based classification models in metabolomic studies

Dietary restriction (DR)-induced changes in the serum metabolome may be biomarkers for physiological status (e.g., relative risk of developing age-related diseases such as cancer). Megavariate analysis (unsupervised hierarchical cluster analysis [HCA]; principal components analysis [PCA]) of serum metabolites reproducibly distinguish DR from ad libitum fed rats. Component-based approaches (i.e., PCA) consistently perform as well as or better than distance-based metrics (i.e., HCA). We therefore tested the following: (A) Do identified subsets of serum metabolites contain sufficient information to construct mathematical models of class membership (i.e., expert systems)? (B) Do component-based metrics out-perform distance-based metrics? Testing was conducted using KNN (k-nearest neighbors, supervised HCA) and SIMCA (soft independent modeling of class analogy, supervised PCA). Models were built with single cohorts, combined cohorts or mixed samples from previously studied cohorts as training sets. Both algorithms over-fit models based on single cohort training sets. KNN models had >85% accuracy within training/test sets, but were unstable (i.e., values of k could not be accurately set in advance). SIMCA models had 100% accuracy within all training sets, 89 % accuracy in test sets, did not appear to over-fit mixed cohort training sets, and did not require post-hoc modeling adjustments. These data indicate that (i) previously defined metabolites are robust enough to construct classification models (expert systems) with SIMCA that can predict unknowns by dietary category; (ii) component-based analyses outperformed distance-based metrics; (iii) use of over-fitting controls is essential; and (iv) subtle inter-cohort variability may be a critical issue for high data density biomarker studies that lack state markers.