Affinity-enhanced protein partitioning in decyl beta-D-glucopyranoside two-phase aqueous micellar systems

Liquid-liquid extraction in two-phase aqueous complex-fluid systems has been proposed as a scalable, versatile, and cost-effective purification method for the downstream processing of biotechnological products. In the case of two-phase aqueous micellar systems, careful choices of the phase-forming surfactants or surfactant mixtures allow these systems to separate biomolecules based on size, hydrophobicity, charge, or specific affinity. In this article, we investigate the affinity-enhanced partitioning of a model affinity-tagged protei--green fluorescent protein fused to a family 9 carbohydrate-binding module (CBM9-GFP)--in a two-phase aqueous micellar system generated from the nonionic surfactant n-decyl beta-D-glucopyranoside (C10G1), which acts simultaneously as the phase-former and the affinity ligand. In this simple system, CBM9-GFP was extracted preferentially into the micelle-rich phase, despite the opposing tendency of the steric, excluded-volume interactions operating between the protein and the micelles. We obtained more than a sixfold increase (from 0.47 to 3.1) in the protein partition coefficient (Kp), as compared to a control case where the affinity interactions were "turned off" by the addition of a competitive inhibitor (glucose). It was demonstrated conclusively that the observed increase in Kp can be attributed to the specific affinity between the CBM9 domain and the affinity surfactant C10G1, suggesting that the method can be generally applied to any CBM9-tagged protein. To rationalize the observed phenomenon of affinity-enhanced partitioning in two-phase aqueous micellar systems, we formulated a theoretical framework to model the protein partition coefficient. The modeling approach accounts for both the excluded-volume interactions and the affinity interactions between the protein and the surfactants, and considers the contributions from the monomeric and the micellar surfactants separately. The model was shown to be consistent with the experimental data, as well as with our current understanding of the CBM9 domain.     

Engineering green fluorescent protein as a dual functional tag

A hexa-histidine (6 x His) sequence was inserted into a surface loop of the green fluorescent protein (GFP) to develop a dual functional GFP useful for both monitoring and purification of recombinant proteins. Two variants (GFP172 and GFP157), differentiated by the site of insertion of the 6xHis sequence, were developed and compared with a control variant (GFPHis) having the 6xHis sequence at its C-terminus. The variants were produced in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). The purification efficiencies by IMAC for all variants were found to be comparable. Purified GFP172 and GFP157 variants retained approximately 60% of the fluorescence compared to that of GFPHis. The reduction in the fluorescence intensity associated with GFP172 and GFP157 was attributed to the lower percentage of fluorescent GFP molecules in these variants. Nonetheless, the rates of fluorescence acquisition were found to be similar for all functional variants. Protein misfolding at an elevated temperature (37 degrees C) was found to be less profound for GFP172 than for GFP157. The dual functional properties of GFP172 were tested with maltose binding protein (MBP) as the fusion partner. The MBP-GFP172 fusion protein remained fluorescent and was purified from E. coli lysate as well as from spiked tobacco leaf extracts in a single-step IMAC. For the latter, a recovery yield of approximately 75% was achieved and MBP-GFP172 was found to coelute with a degraded product of the fusion protein at a ratio of about 4:1. The primary advantage of the chimeric GFP tag having an internal hexa-histidine sequence is that such a tag allows maximum flexibility for protein or peptide fusions since both N- and C-terminal ends of the GFP are available for fusion.     

Strategy for selecting and characterizing linker peptides for CBM9-tagged fusion proteins expressed in Escherichia coli

The influence of linker design on fusion protein production and performance was evaluated when a family 9 carbohydrate-binding module (CBM9) serves as the affinity tag for recombinant proteins expressed in Escherichia coli. Two bioinformatic strategies for linker design were applied: the first identifies naturally occurring linkers within the proteome of the host organism, the second involves screening peptidases and their known specificities using the bioinformatics software MEROPS to design an artificial linker resistant to proteolysis within the host. Linkers designed using these strategies were compared against traditional poly-glycine linkers. Although widely used, glycine-rich linkers were found by tandem MS data to be susceptible to hydrolysis by E. coli peptidases. The natural (PT)(x)P and MEROPS-designed S(3)N(10) linkers were significantly more stable, indicating both strategies provide a useful approach to linker design. Factor X(a) processing of the fusion proteins depended strongly on linker chemistry, with poly(G) and S(3)N(10) linkers showing the fastest cleavage rates. Luminescence resonance energy transfer studies, used to measure average distance of separation between GFP and Tb(III) bound to a strong calcium-binding site of CBM9, revealed that, for a given linker chemistry, the separation distance increases with increasing linker length. This increase was particularly large for poly(G) linkers, suggesting that this linker chemistry adopts a hydrated, extended configuration that makes it particularly susceptible to proteolysis. Differential scanning calorimetry studies on the PT linker series showed that fusion of CBM9 to GFP did not alter the T(m) of GFP but did result in a destabilization, as seen by both a decrease in T(m) and DeltaH(cal), of CBM9. The degree of destabilization increased with decreasing length of the (PT)(x)P linker such that DeltaT(m) = -8.4 degrees C for the single P linker.     

Integrated bioprocessing in Saccharomyces cerevisiae using green fluorescent protein as a fusion partner

In this study, we examine the use of green fluorescent protein (GFP) for monitoring a hexokinase (HXK)-GFP fusion protein in Saccharomyces cerevisiae for various events including expression, degradation, purification, and localization. The fusion, HXK-EK-GFP-6 x His, was constructed where the histidine tag (6 x His) would allow for convenient affinity purification, and the enterokinase (EK) cleavage site would be used for separation of HXK from GFP after affinity purification. Our results showed that both HXK and GFP remained active in the fusion and, more importantly, that there was a linear correlation between HXK activity and GFP fluorescence. Enterokinase cleavage studies revealed that both GFP fluorescence intensity and HXK activity remained unchanged after separation of the fusion proteins, which indicated that fusion of GFP did not cause structural alteration of HXK and thus did not affect the enzymatic activity of HXK. We also found that degradation of the fusion protein occurred, and that degradation was limited to HXK with GFP remaining intact in the fusion. Confocal microscopy studies showed that while GFP was distributed evenly in the yeast cytosol, HXK-GFP fusion followed the correct localization of HXK, which resulted in a di-localization of both cytosol and the nucleus. GFP proved to be a useful fusion partner that may lead to the possibility of integrating the bioprocesses by quantitatively following the entire process visually.     

Characteristics of protein partitioning in an aqueous micellar-gel system

Partitioning of proteins has been studied experimentally in a system combining a gel-bead phase and a nonionic micellar phase. The micellar phase consists of cylindrically shaped micelles, which are completely excluded from the gel-bead phase. Partitioning of single-component protein solutions (myoglobin, ovalbumin, and BSA) is determined by excluded-volume interactions in the micellar phase, and as a result the proteins prefer the gel-bead phase to the micellar phase. The protein concentration inside the gel beads increases with an increase in volume fraction of the micelles and increases with an increase in the size of the proteins. The protein partition coefficients obtained for a binary mixture of myoglobin and bovine serum albumin (BSA) show the same protein concentration dependence as the single-component protein partition coefficients.     

Aqueous two-phase polymer systems as tools for the study of a recombinant surface-expressed Escherichia coli hemagglutinin.

The surface expression of an integral membrane hemagglutinin, HRA1, cloned from Escherichia coli O9: H10:K99 in heterologous E. coli strains was studied by utilizing a variety of polyethylene glycol-dextran and dextran-Ficoll aqueous two-phase polymer systems. Bacteria containing plasmids that encoded the hemagglutinin were found to partition differently from both the host bacteria lacking the plasmid and the original hemagglutinating strain in several of these systems. By using molecular biological techniques, the origin of the partition difference was unambiguously correlated to the expression of HRA1, providing evidence independent of the agglutination phenotype that the protein was accessible to the surrounding milieu. It was demonstrated by using bacterial partition in charge-sensitive systems that the agglutination event was not likely to be due to the presence of a nonspecific positively charged surface protein, as HRA1-expressing clones showed no less affinity for the relatively positive polyethylene glycol-rich upper phase than did control bacteria. This work demonstrates the utility of aqueous polymer two-phase systems for the study of surface-expressed recombinant proteins, due to the sensitivity of the systems and the presence of excellent controls (the host bacteria before plasmid introduction). In cloning and expression studies of surface-associated proteins, two-phase aqueous polymer systems could be used as an alternative to antibody production for the monitoring of surface expression, and these systems may give valuable information on the surface exposure of the protein.     

Establishment of insect cell lines expressing green fluorescent protein on cell surface based on AcMNPV GP64 membrane fusion characteristic

Displaying a protein on the surface of cells has been provided a very successful strategy to function research of exogenous proteins. Based on the membrane fusion characteristic of Autographa californica multiple nucleopolyhedrovirus envelope protein GP64, we amplified and cloned N-terminal signal peptide and C-terminal transmembrane domain as well as cytoplasmic tail domain of gp64 gene into vector pIZ/V5-His with multi-cloning sites to construct the cell surface expression vector pIZ/V5-gp64. To verify that the vector can be used to express proteins on the membrane of insect cells, a recombinant plasmid pIZ/V5-gp64-GFP was constructed by introducing the PCR amplified green fluorescent protein (GFP) gene and transfected into insect cell lines Sf9 and H5. The transected cells were screened with zeocin and cell cloning. PCR verification results showed that the GFP gene was successfully integrated into these cells. Green fluorescence in Sf9-GFP and H5-GFP cells was observed by using confocal laser scanning microscopy and immunofluorescence detection indicated that GFP protein was located on the cell membrane. Western blot results showed that a fusion protein GP64-GFP of about 40 kDa was expressed on the membrane of Sf9-GFP and H5-GFP cells. The expression system constructed in this paper can be used for localization and continuous expression of exogenous proteins on insect cell membrane.     

3',5'-二磷酸核苷酸酶在亲和双水相胶束系统中分配行为研究

以高分子表面活性剂HM-EO为主成相剂,金属螯合表面活性剂Triton X-114-IDA-Cu(Ⅱ)(TX-Cu(Ⅱ))为辅成相剂,构建新型亲和双水相胶束系统(ATPMS)以提高目标产物的萃取选择性,并考察重组蛋白3',5'-二磷酸核苷酸酶(YND)在系统中分配行为。结果表明,系统中不含亲和配基时YND主要分配于胶束缺失相;随着亲和配基含量的增加,YND与TX-Cu(Ⅱ)亲和结合而逐渐分配到胶束富集相并且在系统中显示出优异的稳定性;调节溶液p H能够影响YND亲和分配,最适萃取条件为pH 9.0;增大无机盐浓度,导致更多杂蛋白分配到胶束缺失相,然而对YND分配影响较小。在2.5%HM-EO、0.125%TX-Cu(Ⅱ)、p H 9.0、50 mmol/L Na Cl条件下,实验获得65.8%的酶活回收率。因此亲和ATPMS可以有效用于对富组氨酸蛋白YND的分离纯化,为该体系在重组蛋白的分离纯化试验提供相应的基础依据。     

Combined use of extraction and genetic engineering for protein purification: recovery of beta-galactosidase fused proteins.

Partitioning of beta-galactosidase in aqueous two-phase systems of poly(ethylene glycol) and potassium phosphate is reviewed. The affinity of Escherichia coli beta-galactosidase for the PEG-rich phase dominates also in beta-galactosidase fusion proteins and the concept of using beta-galactosidase as an affinity handle for extraction of other proteins, after fusion, is discussed. A hypothesis is presented, assuming that tryptophan residues at the surface of beta-galactosidase is responsible for its partitioning to the PEG rich phase, and the concept of poly-tryptophan handles fused to the target protein for extraction is introduced.     

LPS removal from an E. coli fermentation broth using aqueous two‐phase micellar system

In biotechnology, endotoxin (LPS) removal from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, as endotoxin is a natural component of bacterial expression systems widely used to manufacture therapeutic proteins. The viability of large‐scale industrial production of recombinant biomolecules of pharmaceutical interest significantly depends on the separation and purification techniques used. The aim of this work was to evaluate the use of aqueous two‐phase micellar system (ATPMS) for endotoxin removal from preparations containing recombinant proteins of pharmaceutical interest, such as green fluorescent protein (GFPuv). Partition assays were carried out initially using pure LPS, and afterwards in the presence of E. coli cell lysate. The ATPMS technology proved to be effective in GFPuv recovery, preferentially into the micelle‐poor phase (K_GFPuv < 1.00), and LPS removal into the micelle‐rich phase (%REM_LPS > 98.00%). Therefore, this system can be exploited as the first step for purification in biotechnology processes for removal of higher LPS concentrations. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Genetic analysis of G protein-coupled receptor expression in Escherichia coli: inhibitory role of DnaJ on the membrane integration of the human central cannabinoid receptor

The overexpression of G protein-coupled receptors (GPCRs) and of many other heterologous membrane proteins in simple microbial hosts, such as the bacterium Escherichia coli, often results in protein mistargeting, aggregation into inclusion bodies or cytoplasmic degradation. Furthermore, membrane protein production is very frequently accompanied by severe cell toxicity. In this work, we have employed a genetic strategy to isolate E. coli mutants that produce markedly increased amounts of the human central cannabinoid receptor (CB1), a pharmacologically significant GPCR that expresses very poorly in wild-type E. coli. By utilizing a CB1 fusion with the green fluorescent protein (GFP) and fluorescence-activated cell sorting (FACS), we screened an E. coli transposon library and identified an insertion in dnaJ that resulted in a large increase in CB1-GFP fluorescence and a dramatic enhancement in bacterial production of membrane-integrated CB1. Furthermore, the dnaJ::Tn5 inactivation suppressed the severe cytotoxicity associated with CB1 production. This revealed an unexpected inhibitory role of the chaperone/ co-chaperone DnaJ in the protein folding or membrane insertion of bacterially produced CB1. Our strategy can be easily adapted to identify expression bottlenecks for different GPCRs or any other integral membrane protein, provide useful and unanticipated mechanistic insights, and assist in the construction of genetically engineered E. coli strains for efficient heterologous membrane protein production.     

Affinity-specific protein separations using ligand-coupled particles in aqueous two-phase systems: I. Process concept and enzyme binding studies for pyruvate kinase and alcohol dehydrogenase from Saccharomyces cerevisiae

A process using ligand-coupled particles in aqueous polyethylene glycol-dextran two-phase polymer systems was developed to achieve a highly selective, scaleable biochemical separation process. Product protein is bound to the ligand-coupled particles that quantitatively distribute to the polyethylene glycol-rich upper phase. Other proteins and contaminants partition preferentially to the dextran-rich lower phase.The process offers significant advantages over affinity partitioning here the ligand is coupled to the backbone of a polyethylene glycol polymer. These advantages include a much wider diversity of ligands that can be coupled to particles and more effective confinement of the ligand in the process. Affinity partition with ligands coupled to particles is more amenable to scale-up than is affinity chromatography. A variety of commercially available Sepharose-based particles are suitable for this process. Homogenates from Saccharomyces cerevisiae, which is genetically altered to overproduce pyruvate kinase, and Cibacron blue F3G-A-coupled Sepharose particles are used as a model system for the process. Binding studies with/without aqueous two-phase systems show that the formation of a two-phase system after the adsorption equilibrium is reached does not affect the apparent dissociation constant. Binding of protein to ligand-coupled particles is more rapid in single-phase systems than in the polymer two-phase system. Single-phase binding eliminates the mass transfer resistance associated with redistribution of product protein from the dextran-rich bottom phase to the polyethylene glycol-rich top phase.     

Glucose-6-phosphate dehydrogenase partitioning in two-phase aqueous mixed (nonionic/cationic) micellar systems

The enzyme glucose-6-phosphate dehydrogenase (G6PD) plays an important role in maintaining the level of NADPH and in producing pentose phosphates for nucleotide biosynthesis. It is also of great value as an analytical reagent, being used in various quantitative assays. In searching for new strategies to purify this enzyme, the partitioning of G6PD in two-phase aqueous mixed (nonionic/cationic) micellar systems was investigated both experimentally and theoretically. Our results indicate that the use of a two-phase aqueous mixed micellar system composed of the nonionic surfactant C(10)E(4) (n-decyl tetra(ethylene oxide)) and the cationic surfactant C(n)TAB (alkyltrimethylammonium bromide, n = 8, 10, or 12) can improve significantly the partitioning behavior of G6PD relative to that obtained in the two-phase aqueous C(10)E(4) micellar system. This improvement can be attributed to electrostatic attractions between the positively charged mixed (nonionic/cationic) micelles and the net negatively charged enzyme G6PD, resulting in the preferential partitioning of G6PD to the top, mixed micelle-rich phase of the two-phase aqueous mixed micellar systems. The effect of varying the cationic surfactant tail length (n = 8, 10, and 12) on the denaturation and partitioning behavior of G6PD in the C(10)E(4) /C(n)TAB/buffer system was investigated. It was found that C(8)TAB is the least denaturing to G6PD, followed by C(10)TAB and C(12)TAB. However, the C(10)E(4)/C(12)TAB/buffer system generated stronger electrostatic attractions with the net negatively charged enzyme G6PD than the C(10)E(4)/C(10)TAB/buffer and the C(10)E(4)/C(8)TAB/buffer systems, when using the same amount of cationic surfactant. Overall, the two-phase aqueous mixed (C(10)E(4)/C(10)TAB) micellar system yielded the highest G6PD partition coefficient of 7.7, with a G6PD yield in the top phase of 71%, providing the optimal balance between the denaturing effect and the electrostatic attractions for the three cationic surfactants examined. A recently developed theoretical framework to predict protein partition coefficients in two-phase aqueous mixed (nonionic/ionic) micellar systems was implemented, and the theoretically predicted G6PD partition coefficients were found to be in reasonable quantitative agreement with the experimentally measured ones.     

Aqueous two-phase metal affinity extraction of heme proteins

An iminodiacetic acid derivative of poly(ethylene glycol) (PEG-IDA) that chelates metal cations has been synthesized and used to extract proteins in metal affinity aqueous two-phase PEG/dextran systems. With less than 1% of the PEG substituted with chelated copper, partition coefficients are shown to increase by factors of up to 37 over extraction with unsubstituted PEG. The proteins studied are preferentially extracted into the Cu(II)PEGIDA phase in proportion to the number of accessible histidine residues on their surface. The affinity contribution to partitioning is proportional to the number of exposed histidine over a very wide range. The partition coefficients of heme-containing proteins measured in the Cu(II)PEG-IDA/dextran systems increase with the pH of the extraction mixture from pH 5.5 to pH 8.0, while partition coefficients in the unsubstituted PEG/dextran systems are very nearly independent of pH. The strong pH dependence of the metalaffinity extraction can be utilized in the recovery of the extracted protein.     

An integral membrane green fluorescent protein marker, Us9-GFP, is quantitatively retained in cells during propidium iodide-based cell cycle analysis by flow cytometry

Previously, we described GFP-spectrin, a membrane-localized derivative of the green fluorescent protein that can be employed as a marker during the simultaneous identification of transfected cells and cell cycle analysis by flow cytometry (Kalejta et al., Cytometry 29: 286-291, 1997). A membrane-anchored GFP fusion protein is necessary because the ethanol permeabilization step required to achieve efficient propidium iodide staining allows cytoplasmic GFP to leach out of the cell. However, viable cells expressing GFP-spectrin are not as bright as cells expressing cytoplasmic GFP and their fluorescence intensity is further diminished after ethanol treatment. Here, we demonstrate that the fluorescence intensity of cells expressing an integral membrane GFP fusion protein (Us9-GFP) is similar to that of cells expressing cytoplasmic GFP and is quantitatively maintained in cells after ethanol treatment. By allowing an accurate assessment of the expression level of GFP, Us9-GFP allows a more precise analysis of the effects of a cotransfected plasmid on the cell cycle and thus represents an improvement upon the original membrane-associated GFP fusion proteins employed in this assay.     

Hydroxypropyl cellulose/poly(ethylene glycol)-co-poly(propylene glycol) aqueous two-phase systems: system characterization and partition of cells and proteins.

Novel aqueous polymeric two-phase systems are described. These systems are formed by mixing hydroxypropyl cellulose (molecular mass 100,000, trade name Klucel L) with poly(ethylene glycol)-co-poly(propylene glycol) copolymer [molecular mass 6,500, poly(propylene glycol) content 50% w/w, trade name Pluronic P105], in a saline buffer. The phase diagram was measured and the interfacial tensions, phase separation times, and lower phase viscosities of three phase systems having constant Pluronic P105 concentration but varying in Klucel L concentration were determined. The partition behavior of a representative cell, bacterium, and protein and the affinity ligand-mediated alteration in the partition behavior of a protein from a yeast extract protein mixture were also characterized. The results suggest that Klucel L/Pluronic P105 phase systems may be cost-effective substitutes for, or complements to, existing aqueous polymeric phase systems. The physical characterization and representative partition data reported here should facilitate application of these new systems.     

Improved imaging of actin filaments in transgenic Arabidopsis plants expressing a green fluorescent protein fusion to the C- and N-termini of the fimbrin actin-binding domain 2

The role of the actin cytoskeleton in plant development is intimately linked to its dynamic behavior. Therefore it is essential to continue refining methods for studying actin organization in living plant cells. The discovery of green fluorescent protein (GFP) has popularized the use of translational fusions of GFP with actin filament (F-actin) side-binding proteins to visualize in vivo actin organization in plants. The most recent of these live cell F-actin reporters are GFP fusions to the actin-binding domain 2 (ABD2) of Arabidopsis fimbrin 1 (ABD2-GFP). To improve ABD2-GFP fluorescence for enhanced in vivo F-actin imaging, transgenic Arabidopsis plants were generated expressing a construct with GFP fused to both the C- and N-termini of ABD2 under the control of the CaMV 35S promoter (35S::GFP-ABD2-GFP). The 35S::GFP-ABD2-GFP lines had significantly increased fluorescence compared with the original 35S::ABD2-GFP lines. The enhanced fluorescence of the 35S::GFP-ABD2-GFP-expressing lines allowed the acquisition of highly resolved images of F-actin in different plant organs and stages of development because of the reduced confocal microscope excitation settings needed for data collection. This simple modification to the ABD2-GFP construct presents an important tool for studying actin function during plant development.     

Tat 蛋白的PTD区段促进GFP蛋白进入骨髓瘤细胞SP2/0

随着生物工程技术的迅速发展 ,多肽与蛋白质类药物的增长速度相当可观 ,可是这些药物常因受到各种因素的影响而疗效偏低 ,其中生物膜的屏障作用是主要因素之一。近年来发现一种来源于人类免疫缺陷病毒ＨＩＶ 1Ｔａｔ(Ｔｒａｎｓ ａｃｔｉｖａ ｔｏｒ)蛋白的蛋白功能区 ,称之为ＰＴＤ区段 (Ｐｒｏｔｅｉｎｔｒａｎｓｄｕｃｔｉｏｎｄｏｍａｉｎ ,ＹＧＲＫＫＲＲＱＲＲＲ)的〔1 ,2〕,能够有效引导肽段或者蛋白质进入细胞 ,具有蛋白传送的功能〔3〕。 1988年Ｍａｕｒｉｃｅ和Ｐａｕｌ发现Ｔａｔ蛋白能够穿过细胞膜〔4〕 ;1994年Ｓｔｅｐｈｅｎ…     

The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins

We present a new streptavidin-binding peptide for both the purification and the detection of recombinant proteins. The peptide possesses nanomolar-affinity for streptavidin and therefore was termed Nano-tag. The Nano-tag(15) is 15 amino acids long and binds to streptavidin with a dissociation constant of 4 nM and the Nano-tag(9) is a 9-mer peptide with a dissociation constant of 17 nM. We demonstrate the one-step purification of Nano-tagged proteins, namely bovine heart fatty acid-binding protein (FABP), bacterial chloramphenicol acetyltransferase (CAT), and green fluorescent protein (GFP), from an in vitro translation system as well as from an Escherichia coli lysate. No significant influence of the Nano-tag(15) and of the conditions during affinity chromatography on maturation or activity of the proteins was observed whereas the Nano-tag(9) revealed a slight decline in the amount and activity of the synthesized proteins. The main advantage of the Nano-tag is the mild and specific elution with washing buffer plus biotin or related compounds, which enables the elution of the bound fusion protein from the streptavidin column in the native state. Additionally, the Nano-tag allowed the detection of recombinant proteins on Western blots by a streptavidin-alkaline phosphatase conjugate.     

A method for three-dimensional protein characterization and its application to a complex plant (corn) extract

Knowledge of host protein properties is critical for developing purification methods for recombinant proteins from a specific host, or for choosing suitable hosts and targeted expression tissues for a specific recombinant protein. A method to obtain a three-dimensional (3D) map (surface hydrophobicity (SH), isoelectric point (pI), and molecular weight (MW)), of a host's aqueous soluble protein properties was developed. The method consists of hydrophobic partitioning in a PEG 3350 (15.7%)-Na(2)SO(4) (8.9%)-NaCl (3%) aqueous two-phase (ATP) system followed by quantitative, 2D-electrophoretic characterization of the proteins of each equilibrium phase and the original extract. The pI and MW of host proteins were obtained directly through 2D electrophoresis. The partition coefficients of individual proteins were obtained by quantitative matching of protein spots in the top and bottom phase gels and calculating the protein partition coefficients from this information. Correlation of the partition coefficient to a SH scale was established by partitioning several model proteins with known surface hydrophobicities in the same ATP system. The inclusion of the extract gel provided for a spot selection criterion based on satisfactory mass balance closure. The method is illustrated by application to a mixture of model proteins and to complex mixtures, that is, corn germ proteins extracted at pH 7 and pH 4.