Genome-scale reconstruction of Salinispora tropica CNB-440 metabolism to study strain-specific adaptation.

The first manually curated genome-scale metabolic model for Salinispora tropica strain CNB-440 was constructed. The reconstruction enables characterization of the metabolic capabilities for understanding and modeling the cellular physiology of this actinobacterium. The iCC908 model was based on physiological and biochemical information of primary and specialised metabolism pathways. The reconstructed stoichiometric matrix consists of 1169 biochemical conversions, 204 transport reactions and 1317 metabolites. A total of 908 structural open reading frames (ORFs) were included in the reconstructed network. The number of gene functions included in the reconstructed network corresponds to 20% of all characterized ORFs in the S. tropica genome. The genome-scale metabolic model was used to study strain-specific capabilities in defined minimal media. iCC908 was used to analyze growth capabilities in 41 different minimal growth-supporting environments. These nutrient sources were evaluated experimentally to assess the accuracy of in silico growth simulations. The model predicted no auxotrophies for essential amino acids, which was corroborated experimentally. The strain is able to use 21 different carbon sources, 8 nitrogen sources and 4 sulfur sources from the nutrient sources tested. Experimental observation suggests that the cells may be able to store sulfur. False predictions provided opportunities to gain new insights into the physiology of this species, and to gap fill the missing knowledge. The incorporation of modifications led to increased accuracy in predicting the outcome of growth/no growth experiments from 76 to 93%. iCC908 can thus be used to define the metabolic capabilities of S. tropica and guide and enhance the production of specialised metabolites.

Cloning, expression and decoding of the cold adaptation of a new widely represented thermolabile subtilisin-like protease.

AIMS: Cloning, expression and characterization of a new cold-adapted protease with potential biotechnological application, isolated from Antarctic bacteria. METHOD AND RESULTS: A subtilisin-like gene was isolated from several Antarctic bacterial genus using CODPEHOP-designed primers and a genome walking method. This gene encodes a precursor protein, which undergoes an autocatalytic cleavage resulting in a 34.6 kDa active cold-adapted protease with a maximum activity at 25-35°C and optimum pH of 8.0-9.0. It showed a higher catalytic efficiency at lower temperatures compared to its mesophilic counterpart. Heat-induced inactivation resulted in a very low melting point. Local packing analysis using the homology model indicated Ala284 as an important cold-adaptation determinant, which was corroborated by the site-directed mutagenesis. CONCLUSIONS: A new thermolabile subtilisin-like protease has been successfully cloned and analysed, and an important hot spot in the evolution of the cold adaptation and substrate specificity of this enzyme was identified and tested. SIGNIFICANCE AND IMPACT OF THE STUDY: This work reports a new cold-adapted protease with a vast representation amongst Antarctic genus, suggesting therefore its evolutionary success in this cold environment. Likewise, important sites for genetic potentiation have been identified, which are extrapolated to other enzymes of the same kind.

Continuous modeling of metabolic networks with gene regulation in yeast and in vivo determination of rate parameters.

A continuous model of a metabolic network including gene regulation to simulate metabolic fluxes during batch cultivation of yeast Saccharomyces cerevisiae was developed. The metabolic network includes reactions of glycolysis, gluconeogenesis, glycerol and ethanol synthesis and consumption, the tricarboxylic acid cycle, and protein synthesis. Carbon sources considered were glucose and then ethanol synthesized during growth on glucose. The metabolic network has 39 fluxes, which represent the action of 50 enzymes and 64 genes and it is coupled with a gene regulation network which defines enzyme synthesis (activities) and incorporates regulation by glucose (enzyme induction and repression), modeled using ordinary differential equations. The model includes enzyme kinetics, equations that follow both mass-action law and transport as well as inducible, repressible, and constitutive enzymes of metabolism. The model was able to simulate a fermentation of S. cerevisiae during the exponential growth phase on glucose and the exponential growth phase on ethanol using only one set of kinetic parameters. All fluxes in the continuous model followed the behavior shown by the metabolic flux analysis (MFA) obtained from experimental results. The differences obtained between the fluxes given by the model and the fluxes determined by the MFA do not exceed 25% in 75% of the cases during exponential growth on glucose, and 20% in 90% of the cases during exponential growth on ethanol. Furthermore, the adjustment of the fermentation profiles of biomass, glucose, and ethanol were 95%, 95%, and 79%, respectively. With these results the simulation was considered successful. A comparison between the simulation of the continuous model and the experimental data of the diauxic yeast fermentation for glucose, biomass, and ethanol, shows an extremely good match using the parameters found. The small discrepancies between the fluxes obtained through MFA and those predicted by the differential equations, as well as the good match between the profiles of glucose, biomass, and ethanol, and our simulation, show that this simple model, that does not rely on complex kinetic expressions, is able to capture the global behavior of the experimental data. Also, the determination of parameters using a straightforward minimization technique using data at only two points in time was sufficient to produce a relatively accurate model. Thus, even with a small amount of experimental data (rates and not concentrations) it was possible to estimate the parameters minimizing a simple objective function. The method proposed allows the obtention of reasonable parameters and concentrations in a system with a much larger number of unknowns than equations. Hence a contribution of this study is to present a convenient way to find in vivo rate parameters to model metabolic and genetic networks under different conditions.

Extension of the selection of protein chromatography and the rate model to affinity chromatography.

The rational selection of optimal protein purification sequences, as well as mathematical models that simulate and allow optimization of chromatographic protein purification processes have been developed for purification procedures such as ion-exchange, hydrophobic interaction and gel filtration chromatography. This paper investigates the extension of such analysis to affinity chromatography both in the selection of chromatographic processes and in the use of the rate model for mathematical modelling and simulation. Two affinity systems were used: Blue Sepharose and Protein A. The extension of the theory developed previously for ion-exchange and HIC chromatography to affinity separations is analyzed in this paper. For the selection of operations two algorithms are used. In the first, the value of η, which corresponds to the efficiency (resolution) of the actual chromatography and, Σ, which determines the amount of a particular contaminant eliminated after each separation step, which determines the purity, have to be determined. It was found that the value of both these parameters is not generic for affinity separations but will depend on the type of affinity system used and will have to be determined on a case by case basis. With Blue Sepharose a salt gradient was used and with Protein A, a pH gradient. Parameters were determined with individual proteins and simulations of the protein mixtures were done. This approach allows investigation of chromatographic protein purification in a holistic manner that includes ion-exchange, HIC, gel filtration and affinity separations for the first time.

Viral vectors for the treatment of alcoholism: use of metabolic flux analysis for cell cultivation and vector production.

The HEK293 cell line has been used for the production of adenovirus vectors to be used in the potential treatment of alcoholism using a gene therapy strategy. Culture optimization and scale-up has been achieved by first adapting the cells to serum-free media and secondly by growing them in suspension. Adenovirus production after infection was increased, resulting in higher specific glucose consumption and lactate accumulation rates compared to the growth phase. We applied media design tools and Metabolic Flux Analysis (MFA) to compare the metabolic states of cells during growth and adenovirus production and to optimize culture media according to the metabolic demand of the cells in terms of glucose and glutamine concentrations. This allowed obtaining a higher maximum cell concentration and increased adenovirus production by minimizing the production of metabolites that can have an inhibitory effect on cell growth. We have proposed a stoichiometric equation for adenovirus synthesis. MFA results allowed determination of how these changes in composition affected the way cells distribute their nutrient resources during cell growth and virus production. Virus purification was successfully achieved using chromatography and Aqueous Two-Phase Systems (ATPS).

Stoichiometric model and metabolic flux analysis for Leptospirillum ferrooxidans.

A metabolic model for Leptospirillum ferrooxidans was developed based on the genomic information of an analogous iron oxidizing bacteria and on the pathways of ferrous iron oxidation, nitrogen and CO(2) assimilation based on experimental evidence for L. ferrooxidans found in the literature. From this metabolic reconstruction, a stoichiometric model was built, which includes 86 reactions describing the main catabolic and anabolic aspects of its metabolism. The model obtained has 2 degrees of freedom, so two external fluxes were estimated to achieve a determined and observable system. By using the external oxygen consumption rate and the generation flux biomass as input data, a metabolic flux map with a distribution of internal fluxes was obtained. The results obtained were verified with experimental data from the literature, achieving a very good prediction of the metabolic behavior of this bacterium at steady state.

A stoichiometric model of Acidithiobacillus ferrooxidans ATCC 23270 for metabolic flux analysis.

A stoichiometric model of Acidithiobacillus ferrooxidans based on the sequenced genome from strain ATCC 23270 is derived and parameterized using genome/pathway databases. The model describes the main aspects of catabolism and anabolism. By the construction and utilization of the mathematical determination of the network, metabolic flux analysis is performed for such a bacterium for the first time and results are successfully verified by comparison to literature values. This first metabolic model of A. ferrooxidans is able to simulate the main aspects of metabolism and will be useful for further investigation and improvement of bioleaching procedures.

A discrete mathematical model applied to genetic regulation and metabolic networks.

This paper describes the use of a discrete mathematical model to represent the basic mechanisms of regulation of the bacteria E. coli in batch fermentation. The specific phenomena studied were the changes in metabolism and genetic regulation when the bacteria use three different carbon substrates (glucose, glycerol, and acetate). The model correctly predicts the behavior of E. coli vis-à-vis substrate mixtures. In a mixture of glucose, glycerol, and acetate, it prefers glucose, then glycerol, and finally acetate. The model included 67 nodes; 28 were genes, 20 enzymes, and 19 regulators/biochemical compounds. The model represents both the genetic regulation and metabolic networks in an inrtegrated form, which is how they function biologically. This is one of the first attempts to include both of these networks in one model. Previously, discrete mathematical models were used only to describe genetic regulation networks. The study of the network dynamics generated 8 (2(3)) fixed points, one for each nutrient configuration (substrate mixture) in the medium. The fixed points of the discrete model reflect the phenotypes described. Gene expression and the patterns of the metabolic fluxes generated are described accurately. The activation of the gene regulation network depends basically on the presence of glucose and glycerol. The model predicts the behavior when mixed carbon sources are utilized as well as when there is no carbon source present. Fictitious jokers (Joker1, Joker2, and Repressor SdhC) had to be created to control 12 genes whose regulation mechanism is unknown, since glycerol and glucose do not act directly on the genes. The approach presented in this paper is particularly useful to investigate potential unknown gene regulation mechanisms; such a novel approach can also be used to describe other gene regulation situations such as the comparison between non-recombinant and recombinant yeast strain, producing recombinant proteins, presently under investigation in our group.

Characterization of Ferroplasma acidiphilum growing in pure and mixed culture with Leptospirillum ferriphilum.

Biomining is defined as biotechnology for metal recovery from minerals, and is promoted by the concerted effort of a consortium of acidophile prokaryotes, comprised of members of the Bacteria and Archaea domains. Ferroplasma acidiphilum and Leptospirillum ferriphilum are the dominant species in extremely acid environments and have great use in bioleaching applications; however, the role of each species in this consortia is still a subject of research. The hypothesis of this work is that F. acidiphilum uses the organic matter secreted by L. ferriphilum for growth, maintaining low levels of organic compounds in the culture medium, preventing their toxic effects on L. ferriphilum. To test this hypothesis, a characterization of Ferroplasma acidiphilum strain BRL-115 was made with the objective of determining its optimal growth conditions. Subsequently, under the optimal conditions, L. ferriphilum and F. acidiphilum were tested growing in each other's supernatant, in order to define if there was exchange of metabolites between the species. With these results, a mixed culture in batch cyclic operation was performed to obtain main specific growth rates, which were used to evaluate a mixed metabolic model previously developed by our group. It was observed that F. acidiphilum, strain BRL-115 is a chemomixotrophic organism, and its growth is maximized with yeast extract at a concentration of 0.04% wt/vol. From the experiments of L. ferriphilum growing on F. acidiphilum supernatant and vice versa, it was observed that in both cases cell growth is favorably affected by the presence of the filtered medium of the other microorganism, proving a synergistic interaction between these species. Specific growth rates were obtained in cyclic batch operation of the mixed culture and were used as input data for a Flux Balance Analysis of the mixed metabolic model, obtaining a reasonable behavior of the metabolic fluxes and the system as a whole, therefore consolidating the model previously developed. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1390-1396, 2016.

Ultrasonographic display of complex vascular rings.

Ultrasonographic display of vascular ring anatomy has been limited to single-plane views. This does not readily allow for a three-dimensional interpretation of structural relations. A method that included a sweep consisting of multiple contiguous frontal planes was used in 12 patients with a vascular ring before repair for the evaluation of the arch sidedness and number, brachiocephalic vessel pattern, upper descending thoracic aorta sidedness, ductus arteriosus site or sites and proximal pulmonary arteries; 7 patients had Doppler color flow imaging. Complete imaging of the luminal vascular components was possible in all but one patient. In four other patients, atretic segments of the vascular ring could not be displayed. The addition of Doppler color flow imaging especially aided in the tracing of multiple vascular structures in complex cases and in assessing ductus arteriosus and arch patency. The use of a suprasternal frontal sweep with posterior angulation could display encirclement of the air-filled trachea. Vascular ring segments without lumens could not be displayed.

Two-dimensional and Doppler echocardiographic assessment of neonatal arterial repair for transposition of the great arteries.

The arterial switch procedure has become an accepted reparative technique for transposition of the great arteries with or without ventricular septal defect. In this study the accuracy of prospective noninvasive imaging in detecting arterial tract obstruction and the prevalence and severity of arterial valvular regurgitation (as assessed by Doppler ultrasound) were evaluated in survivors of arterial repair. All 53 study patients underwent two-dimensional echocardiographic examination 2 days to 20 months (median 7 months) postoperatively; 43 patients also had pulsed and continuous wave Doppler studies. The accuracy of the noninvasive evaluation of arterial tract obstruction was determined by comparison of Doppler maximal instantaneous gradients with peak to peak gradients at nonsimultaneous catheterization in 26 patients. Twenty-one (81%) of the 26 patients underwent catheterization and successful pulsed and continuous wave Doppler examination of the right heart; 17 (81%) of these 21 had a maximal pressure gradient within 20 mm Hg of the peak to peak gradient obtained at catheterization. Echocardiographic identification of the stenotic site was correct in all eight of the patients in this group requiring reoperation. Twenty-three (88%) of the 26 patients who underwent catheterization had successful Doppler interrogation of the aortic tract; 22 (96%) of these 23 had a maximal instantaneous gradient within 20 mm Hg of the peak to peak catheterization gradient. Fourteen (32%) of 43 patients had mild or moderate pulmonary regurgitation by Doppler study. Three (7%) of the 43 had mild aortic regurgitation.

Stoichiometric model and flux balance analysis for a mixed culture of Leptospirillum ferriphilum and Ferroplasma acidiphilum.

The oxidation process of sulfide minerals in natural environments is achieved by microbial communities from the Archaea and Bacteria domains. A metabolic reconstruction of two dominant species, Leptospirillum ferriphilum and Ferroplasma acidiphilum, which are always found together as a mixed culture in this natural environments, was made. The metabolic model, composed of 152 internal reactions and 29 transport reactions, describes the main interactions between these species, assuming that both use ferrous iron as energy source, and F. acidiphilum takes advantage of the organic compounds secreted by L. ferriphilum for chemomixotrophic growth. A first metabolic model for a mixed culture used in bacterial leaching is proposed in this article, which pretends to represent the characteristics of the mixed culture in a simplified manner. It was evaluated with experimental data through flux balance analysis (FBA) using as objective function the maximization of biomass. The growth yields on ferrous iron obtained for each microorganism are consistent with experimental data, and the flux distribution obtained allows understanding of the metabolic capabilities of both microorganisms growing together in a bioleaching process. The model was used to simulate the growth of F. acidiphilum on different substrates, to determine in silico which compounds maximize cell growth, and which are essential. Knockout simulations were carried out for L. ferriphilum and F. acidiphilum metabolic models, predicting key enzymes of central metabolism. The results of this analysis are consistent with experimental data from literature, showing a robust behavior of the metabolic model.

Analyzing redox balance in a synthetic yeast platform to improve utilization of brown macroalgae as feedstock.

Macroalgae have high potential to be an efficient, and sustainable feedstock for the production of biofuels and other more valuable chemicals. Attempts have been made to enable the co-fermentation of alginate and mannitol by Saccharomyces cerevisiae to unlock the full potential of this marine biomass. However, the efficient use of the sugars derived from macroalgae depends on the equilibrium of cofactors derived from the alginate and mannitol catabolic pathways. There are a number of strong metabolic limitations that have to be tackled before this bioconversion can be carried out efficiently by engineered yeast cells. An analysis of the redox balance during ethanol fermentation from alginate and mannitol by Saccharomyces cerevisiae using metabolic engineering tools was carried out. To represent the strain designed for conversion of macroalgae carbohydrates to ethanol, a context-specific model was derived from the available yeast genome-scale metabolic reconstructions. Flux balance analysis and dynamic simulations were used to determine the flux distributions. The model indicates that ethanol production is determined by the activity of 4-deoxy-l-erythro-5-hexoseulose uronate (DEHU) reductase (DehR) and its preferences for NADH or NADPH which influences strongly the flow of cellular resources. Different scenarios were explored to determine the equilibrium between NAD(H) and NADP(H) that will lead to increased ethanol yields on mannitol and DEHU under anaerobic conditions. When rates of mannitol dehydrogenase and DehRNADH tend to be close to a ratio in the range 1-1.6, high growth rates and ethanol yields were predicted. The analysis shows a number of metabolic limitations that are not easily identified through experimental procedures such as quantifying the impact of the cofactor preference by DEHU reductase in the system, the low flux into the alginate catabolic pathway, and a detailed analysis of the redox balance. These results show that production of ethanol and other chemicals can be optimized if a redox balance is achieved. A possible methodology to achieve this balance is presented. This paper shows how metabolic engineering tools are essential to comprehend and overcome this limitation.

The ontogeny of serum GH binding protein in man: a possible indicator of hepatic GH receptor development.

Experiments in rabbits suggest that a serum GH binding protein (GH-BP) probably is derived largely from hepatic membrane bound GH receptors. Human serum contains a specific GH binding protein which can be easily measured by incubation with [125I] hGH and separation of [125I] hGH-BP complexes from free [125I] hGH by gel filtration through an Ultrogel AcA 44 minicolumn. In each assay GH-BP activity of a reference (normal young adult) serum is similarly run and the results are expressed as a percentage of the activity of the unknown serum divided by the GH-BP activity of the reference serum after correction for the expected inhibition of GH binding resulting from the GH content of the unknown serum. The mean relative specific GH binding protein (RSGH-BP) of cord serum from 11 premature infants was only 3.2 +/- 1.4% (SE) and of cord serum from 17 full term infants was 14.9 +/- 2.5%. During the first two decades of life there was a progressive rise of RSGH-BP with considerable individual variation. The mean serum RSGH-BP of 13 such subjects was 54.5 +/- 6.2%. More uniform RSGH-BP results were obtained in serum from 15 young adults, 91.7 +/- 7.4%. Lower RSGH-BP 77.2 +/- 5.4% was found in serum from 12 healthy older adults (age 60 to 70 years). The low levels of RSGH-BP in fetal serum are consistent with the reported low concentrations of GH receptors in sheep and rat fetal liver membranes. We suggest the measurement of GH-BP activity provides a simple, noninvasive measure of the ontogeny of GH receptors of human beings.

Role of early postoperative surface echocardiography in the pediatric cardiac intensive care unit.

OBJECTIVE: To compare surface echocardiographic data with catheterization and surgical observation as a way of deciding on the need to reoperate to correct hemodynamically important sequelae following pediatric cardiac surgery; to determine the false-negative diagnosis rate of surface echocardiography. DESIGN: Case series. SETTING: Tertiary-care center, pediatric cardiac intensive care unit. PATIENTS: All 39 patients who underwent reoperation because of hemodynamically significant anatomic sequelae following primary or elective secondary surgery in 1 calendar year. INTERVENTIONS: None. MEASUREMENTS: Two-dimensional and color Doppler ultrasound assessment of anatomy and physiology following cardiac surgery. RESULTS: In 85 percent, surface echocardiography provided sufficient information for surgeons to reoperate on the same admission. Detection of important residual shunts or arterial stenoses and identification of anatomic causes of pulmonary undercirculation (or overcirculation) in palliated single ventricle are feasible. CONCLUSION: Early postoperative surface echocardiography is a viable way to decide on the hemodynamic adequacy of cardiac surgery.

Rational design of purification processes for recombinant proteins.

Recent developments in the rational design of purification processes for recombinant proteins are discussed. A review of the main issues involved in process design for protein separation and purification is presented with particular emphasis on the challenges posed by recombinant proteins. This includes physicochemical characterization of target protein and main contaminants, the use of rigorous mathematical modeling and process simulation as well as the development of an expert system and the application of this technology for optimization and design of large scale processes. An expert system for selection of optimal protein separation sequences will give the user a number of alternatives chosen on the basis of extensive data back-up on proteins and unit operations.

Design of enzyme systems for selective product release from microbial cells; isolation of a recombinant protein from yeast.

The development of expression systems for recombinant proteins and recombinant protein particles that cannot be secreted and that are located in specific cell locations necessitates the development of novel, more selective, techniques for cell disruption. Mechanical cell disruption methods do not discriminate the release of the desired product from among a host of other contaminating molecules and cell debris, and they also may damage the protein product. In contrast, the use of lytic enzyme systems, which can provide biological specificity to the process of cell lysis and disruption, shows an interesting potential for controlled lysis. In this report, the design and the use of lytic enzyme systems for differential product release from microbial cells have been reviewed. Lytic enzyme systems are usually specific either for yeast or for different types of bacteria. Moreover, the activity profile of a lytic system will have an effect on the product distribution. This profile can be manipulated at the genetic, physiological, production reactor, enzyme purification, and lysis reactor levels. Alternative process designs that will allow the sequential release of products from different cell locations have been reviewed and discussed. Alternatives have been explored by process modeling, process simulation, and optimization techniques. These studies show that the use of lytic enzyme systems has tremendous promise as a method of controlled lysis and differential product release. Finally, the release of a specific recombinant protein, human serum albumin (HSA) from yeast cells, has been investigated. The low levels of wall-lytic protease present in the Oerskovia lytic enzyme system have no deleterious effect on the protein product, and the level of HSA extracted from two positive yeast clones using lytic enzymes is similar to that extracted after bead breakage.

Changes in ventricular geometry early after Fontan operation.

Acute removal of a ventricular volume overload, in the face of relatively unchanging ventricular mass, results in geometric alterations that can impair diastolic ventricular performance after Fontan operation. We investigated whether geometric alterations were (1) more severe after Fontan operation than after hemi-Fontan operation, (2) more severe in infants than in children, and (3) more severe in the morphologic right ventricle than in the morphologic left ventricle. We studied 22 patients, 11 with hypoplastic left heart syndrome and 11 with a functionally single morphologic left ventricle. After Fontan operation, right ventricular end-diastolic volume declined by 52% from 36.6 +/- 12.2 to 17.5 +/- 9.3 mL. Right ventricular wall thickness increased from 6.8 +/- 1.3 to 7.5 +/- 1.8 mm. Left ventricular end-diastolic volume diminished by 40% from 57.6 +/- 23.3 to 34.8 +/- 17.6 mL. Left ventricular wall thickness increased from 7.0 +/- 2.1 to 8.7 +/- 1.9 mm. The alterations in right ventricular geometry are more marked early after Fontan operation than early after the hemi-Fontan procedure. Geometric alterations in the left ventricle appear to be more severe in infants than in children. Finally, alterations in the morphologic right ventricle appear to be of similar magnitude to those in the morphologic left ventricle.

Metabolic control analysis of monoclonal antibody synthesis.

A general route for protein synthesis in eukaryotic cells has been proposed and applied to monoclonal antibody (MAb) synthesis. It takes into account transcription of the gene, binding of ribosomes to mRNA, and polypeptide elongation including binding to SRP (signal recognition particles) and SRP-receptor, competing translocation, folding and glycosylation, assembly of the heavy and light chains in a tetrameric protein and Golgi processing and secretion. A comprehensive model was built on the basis of the proposed pathway. The model takes into account the mechanism of each step. Metabolic control analysis (MCA) principles were applied to the general pathway using the proposed model, and control coefficients were calculated. The results show a shared flux control (of both pathway flux and flux ratio at the branch) among different steps, i.e., transcription, folding, glycosylation, translocation and building blocks synthesis. The steps sharing the control depend on the concentration of building blocks, pathway flux and levels of OST (oligosacharyl transferase), BiP (heavy chain binding protein) and PDI (protein disulfide isomerase). Model predictions compare well with experimental data for MAb synthesis, explaining the control structure of the route and the heterogeneity of the product and also addressing future targets for improvement of the production rate of MAbs.

Effect of electrostatic energy on partitioning of proteins in aqueous two-phase systems.

An attempt has been made to adopt a different approach to evaluate the effect of a protein's charge on its partitioning behaviour in PEG/salt aqueous two-phase systems (ATPS). This has been done using a computer methodology (DelPhi) that allows the calculation of the electrostatic solvation energy that charged proteins present in a particular media such as aqueous polymer-salt systems. This calculation was done for the protein in each of the phases and a correlation was investigated that related the electrostatic energy difference of the protein in each of the phases and its partition coefficient in ATPS. Such correlation resulted in a statistical model that also included the effect of molecular weight and a shape factor at each particular pH. A global correlation which included the effect of pH was also found. All the correlations were statistically evaluated and gave good results.