Structure, Specificity, and Mode of Interaction for Bacterial Albumin-binding Modules

The Journal of Biological Chemistry. Mar, 2002  |  Pubmed ID: 11751858

We have determined the solution structure of an albumin binding domain of protein G, a surface protein of group C and G streptococci. We find that it folds into a left handed three-helix bundle similar to the albumin binding domain of protein PAB from Peptostreptococcus magnus. The two domains share 59% sequence identity, are thermally very stable, and bind to the same site on human serum albumin. The albumin binding site, the first determined for this structural motif known as the GA module, comprises residues spanning the first loop to the beginning of the third helix and includes the most conserved region of GA modules. The two GA modules have different affinities for albumin from different species, and their albumin binding patterns correspond directly to the host specificity of C/G streptococci and P. magnus, respectively. These studies of the evolution, structure, and binding properties of the GA module emphasize the power of bacterial adaptation and underline ecological and medical problems connected with the use of antibiotics.

Mutational Analysis of the Interaction Between Albumin-binding Domain from Streptococcal Protein G and Human Serum Albumin

Protein Science : a Publication of the Protein Society. Feb, 2002  |  Pubmed ID: 11790830

Streptococcal protein G (SpG) is a bacterial cell surface receptor exhibiting affinity to both human immunoglobulin (IgG) and human serum albumin (HSA). Interestingly, the serum albumin and immunoglobulin-binding activities have been shown to reside at functionally and structurally separated receptor domains. The binding domain of the HSA-binding part has been shown to be a 46-residue triple alpha-helical structure, but the binding site to HSA has not yet been determined. Here, we have investigated the precise binding region of this bacterial receptor by protein engineering applying an alanine-scanning procedure followed by binding studies by surface plasmon resonance (SPR). The secondary structure as well as the HSA binding of the resulting albumin-binding domain (ABD) variants were analyzed using circular dichroism (CD) and affinity blotting. The analysis shows that the HSA binding involves residues mainly in the second alpha-helix.

Strategy for Highly Selective Ion-exchange Capture Using a Charge-polarizing Fusion Partner

Journal of Chromatography. A. Jan, 2002  |  Pubmed ID: 11822381

To achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography, a novel basic protein domain is used as a purification handle. The proteolytic instability usually encountered for basic peptide tags is avoided by the use of a highly constrained alpha-helical domain based on staphylococcal protein A into which positively charged amino acids have been introduced. Here we show that this domain, consisting of 58 amino acids with a calculated isoelectric point (pI) of 10.5, can be used to efficiently capture different fused target proteins, such as a bacterial DNA polymerase (Klenow fragment), a viral protease (3C) and a fungal lipase (Cutinase). In contrast to standard cation-exchange chromatography, efficient capture can be achieved also at a pH value higher than the pI of the fusion protein, demonstrated here by Zbasic-Klenow polymerase (pI approximately/= 5.8) and ZZ-Cutinase-Zbasic (pI approximately/= 7.2) both purified at a pH of 7.5. These results show that the Zbasic domain is able to confer a regional concentration of positive charge on the fusion protein even at a relatively high pH. Hence, the data suggest that this domain could be used for highly efficient and selective capture of target proteins at conditions where most host-cell proteins do not bind to the chromatographic resin. The obtained purity after this one-step procedure suggests that the strategy could be an alternative to standard affinity chromatography. Methods for site-specific proteolysis of the fusion proteins to release native target proteins are also discussed.

Integrated Strategy for Selective Expanded Bed Ion-exchange Adsorption and Site-specific Protein Processing Using Gene Fusion Technology

Journal of Biotechnology. Jun, 2002  |  Pubmed ID: 12142146

The highly charged domain Z(basic) can be used as a fusion partner to enhance adsorption of target proteins to cation exchanging resins at high pH-values. In this paper, we describe a strategy for purification of target proteins fused to Z(basic) at a constant physiological pH using cation exchange chromatography in an expanded bed mode. We show that two proteins, Klenow DNA polymerase and the viral protease 3C, can be efficiently purified from unclarified Escherichia coli homogenates in a single step with a selectivity analogous to what is normally achieved by affinity chromatography. The strategy also includes an integrated site-specific removal of the Z(basic) purification handle to yield a free target protein.

Escherichia Coli Single-stranded DNA-binding Protein, a Molecular Tool for Improved Sequence Quality in Pyrosequencing

Electrophoresis. Sep, 2002  |  Pubmed ID: 12373756

Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technique based on a DNA sequencing by synthesis principle. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis. In order to expand the field for pyrosequencing, the read length needs to be improved and efforts have been made to purify reaction components as well as add single-stranded DNA-binding protein (SSB) to the pyrosequencing reaction. In this study, we have performed a systematic effort to analyze the effects of SSB by comparing the pyrosequencing result of 103 independent complementary DNA (cDNA) clones. More detailed information about the cause of low quality sequences on templates with different characteristics was achieved by thorough analysis of the pyrograms. Also, real-time biosensor analysis was performed on individual cDNA clones for investigation of primer annealing and SSB binding on these templates. Results from these studies indicate that templates with high performance in pyrosequencing without SSB possess efficient primer annealing and low SSB affinity. Alternative strategies to improve the performance in pyrosequencing by increasing the primer-annealing efficiency have also been evaluated.

Engineering Streptococcal Protein G for Increased Alkaline Stability

Protein Engineering. Oct, 2002  |  Pubmed ID: 12468718

Most protein-based affinity chromatography media are very sensitive towards alkaline treatment, which is a preferred method for regeneration and removal of contaminants from the purification devices in industrial applications. In a previous study, we concluded that a simple and straightforward strategy consisting of replacing asparagine residues could improve the stability towards alkaline conditions. In this study, we have shown the potential of this rationale by stabilizing an IgG-binding domain of streptococcal protein G, i.e. the C2 domain. In order to analyze the contribution of the different amino acids to the alkaline sensitivity of the domain we used a single point mutation strategy. Amino acids known to be susceptible towards high pH, asparagine and glutamine, were substituted for less-alkali-susceptible residues. In addition, aspartic acid residues were mutated to evaluate if the stability could be further increased. The stability of the different C2 variants was subsequently analyzed by exposing them to NaOH. The obtained results reveal that the most sensitive amino acid towards alkaline conditions in the structure of C2 is Asn36. The double mutant, C2(N7,36A), was found to be the most stable mutant constructed. In addition to the increased alkaline stability and also very important for potential use as an affinity ligand, this mutated variant also retains the secondary structure, as well as the affinity to the Fc fragment of IgG.

Methylotrophic Yeast Pichia Pastoris As a Host for Production of ATP-diphosphohydrolase (apyrase) from Potato Tubers (Solanum Tuberosum)

Protein Expression and Purification. Feb, 2003  |  Pubmed ID: 12597881

ATP-diphosphohydrolase (apyrase) catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside tri- and di-phosphates in the presence of divalent cations. This enzyme has broad substrate specificity for nucleotides, which makes it an ideal enzyme for different biotechnical applications, such as DNA sequencing and platelet-aggregation inhibition. The only commercially available apyrase is isolated from potato tubers. To avoid batch-to-batch variations in activity and quality, we decided to produce a recombinant enzyme. The methylotrophic yeast Pichia pastoris was chosen as an eukaryotic expression host. The coding sequence of potato apyrase, without the signal peptide, was cloned into the YpDC541 vector to create a fusion with the alpha-mating secretion signal of Saccharomyces cerevisiae. The gene was placed under the control of the methanol-inducible alcohol oxidase promoter. The YpDC541-apyrase construct was integrated into P. pastoris strain SMD1168. Methanol induction resulted in secretion of apyrase to a level of 1mg/L. The biologically active recombinant apyrase was purified by hydrophobic interaction and ion exchange chromatography. According to SDS-PAGE and Western blot analysis, the purified enzyme showed to be hyperglycosylated. By enzymatic removal of N-glycans, a single band corresponding to a molecular mass of 48kDa was detected. The recombinant apyrase was found to function well when it was used in combination with the Pyrosequencing technology.

An Improved Dual-expression Concept, Generating High-quality Antibodies for Proteomics Research

Biotechnology and Applied Biochemistry. Dec, 2003  |  Pubmed ID: 12875650

A novel, improved dual bacterial-expression system, designed for large-scale generation of high-quality polyclonal antibody preparations intended for proteomics research, is presented. The concept involves parallel expression of cDNA-encoded proteins, as a fusion with two different tags in two separate vector systems. Both systems enable convenient blotting procedures for expression screening on crude bacterial cell cultures and single-step affinity purification under denaturing conditions. One of the fusion proteins is used to elicit antibodies, and the second fusion protein is used in an immobilized form as an affinity ligand to enrich antibodies with selective reactivity to the cDNA-encoded part, common for the two fusion proteins. To evaluate the system, four cDNA clones from putative nuclear proteins from the non-biting midge Chironomus tentans were expressed. Antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting and immunofluorescence procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The four antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the four antibodies gave specific staining in Western-blot analysis of nuclear cell extracts. Furthermore, two of the antibody preparations gave specific staining in immunofluorescence analysis of C. tentans cells. We conclude that the dual-vector concept presented offers a highly stringent strategy for the generation of monospecific polyclonal antibodies, which are useful in proteomics research.

Evaluation of Different Linker Regions for Multimerization and Coupling Chemistry for Immobilization of a Proteinaceous Affinity Ligand

Protein Engineering. Dec, 2003  |  Pubmed ID: 14983098

Alkaline conditions are generally preferred for sanitization of chromatography media by cleaning-in-place (CIP) protocols in industrial biopharmaceutical processes. The use of such rigorous conditions places stringent demands on the stability of ligands intended for use in affinity chromatography. Here, we describe efforts to meet these requirements for a divalent proteinaceous human serum albumin (HSA) binding ligand, denoted ABD*dimer. The ABD*dimer ligand was constructed by genetic head-to-tail linkage of two copies of the ABD* moiety, which is a monovalent and alkali-stabilized variant of one of the serum albumin-binding motifs of streptococcal protein G. Dimerization was performed to investigate whether a higher HSA-binding capacity could be obtained by ligand multimerization. We also investigated the influence on alkaline stability and HSA-binding capacity of three variants (VDANS, VDADS and GGGSG) of the inter-domain linker. Biosensor binding studies showed that divalent ligands coupled using non-directed chemistry demonstrate an increased molar HSA-binding capacity compared with monovalent ligands. In contrast, equal molar binding capacities were observed for both types of ligands when using directed ligand coupling chemistry involving the introduction and recruitment of a unique C-terminal cysteine residue. Significantly higher molar binding capacities were also detected when using the directed coupling chemistry. These results were confirmed in affinity chromatography binding capacity experiments, using resins containing thiol-coupled ligands. Interestingly, column sanitization studies involving exposure to 0.1 M NaOH solution (pH 13) showed that of all the tested constructs, including the monovalent ligand, the divalent ligand construct containing the VDADS linker sequence was the most stable, retaining 95% of its binding capacity after 7 h of alkaline treatment.

Improving the Tolerance of a Protein a Analogue to Repeated Alkaline Exposures Using a Bypass Mutagenesis Approach

Proteins. May, 2004  |  Pubmed ID: 15048831

Staphylococcal protein A (SPA) is a cell surface protein expressed by Staphylococcus aureus. It consists of five repetitive domains. The five SPA-domains show individual interaction to the Fc-fragment as well as certain Fab-fragments of immunoglobulin G (IgG) from most mammalian species. Due to the high affinity and selectivity of SPA, it has a widespread use as an affinity ligand for capture and purification of antibodies. One of the problems with proteinaceous affinity ligands in large-scale purification is their sensitivity to alkaline conditions. SPA however, is considered relatively stable to alkaline treatment. Nevertheless, it is desirable to further improve the stability in order to enable an SPA-based affinity medium to withstand even longer exposure to the harsh conditions associated with cleaning-in-place (CIP) procedures. For this purpose, a protein engineering strategy, which was used earlier for stabilization and consists of replacing the asparagine residues, is employed. Since Z in its "nonengineered" form already has a significant tolerance to alkaline treatment, small changes in stability due to the mutations are difficult to assess. Hence, in order to enable detection of improvements regarding the alkaline resistance of the Z domain, we chose to use a bypass mutagenesis strategy using a mutated variant Z(F30A) as a surrogate framework. Z(F30A) has earlier been shown to possess an affinity to IgG that is similar to the wild-type but also demonstrates decreased structural stability. Since the contribution of the different asparagine residues to the deactivation rate of a ligand is dependent on the environment and also the structural flexibility of the particular region, it is important to consider all sensitive amino acids one by one. The parental Z-domain contains eight asparagine residues, each with a different impact on the alkaline stability of the domain. By exchanging asparagine 23 for a threonine, we were able to increase the stability of the Z(F30A) domain in alkaline conditions. Also, when grafting the N23T mutation to the Z scaffold, we were able to detect an increased tolerance to alkaline treatment compared to the native Z molecule.

Toward Pyrosequencing on Surface-attached Genetic Material by Use of DNA-binding Luciferase Fusion Proteins

Analytical Biochemistry. Jun, 2004  |  Pubmed ID: 15136162

Mutation detection and single-nucleotide polymorphism genotyping require screening of large samples of materials and therefore the importance of high-throughput DNA analysis techniques is significant. Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technology based on the sequencing-by-synthesis principle. Currently, the technique is limited to simultaneous analysis of 96 or 384 samples. Earlier, attempts to increase the sample capacity were made using micromachined filter chamber arrays where parallel analyses of nanoliter samples could be monitored in real time. We have developed a strategy for specific immobilization of the light-producing enzyme luciferase to the DNA template within a reaction chamber. By this approach, luciferase is genetically fused to a DNA-binding protein (Klenow polymerase or Escherichia coli single-stranded DNA-binding (SSB) protein) and to a purification handle (Z(basic)). The proteins are produced in E. coli and purified using cation and anion exchange chromatography with removal of Z(basic). The produced proteins have been analyzed using an assay for complete primer extension of DNA templates immobilized on magnetic beads detected by pyrosequencing chemistry. Results from these experiments show that the proteins bind selectively to the immobilized DNA and that their enzymatic domains were active. Z(basic)-SSB-luciferase produced the highest signal in this assay and was further exploited as enzymatic reagent for DNA sequencing.

Genome-based Proteomics

Electrophoresis. May, 2004  |  Pubmed ID: 15174050

Protein-protein interactions play crucial roles in various biological pathways and functions. Therefore, the characterization of protein levels and also the network of interactions within an organism would contribute considerably to the understanding of life. The availability of the human genome sequence has created a range of new possibilities for biomedical research. A crucial challenge is to utilize the genetic information for better understanding of protein distribution and function in normal as well as in pathological biological processes. In this review, we have focused on different platforms used for systematic genome-based proteome analyses. These technologies are in many ways complementary and should be seen as various ways to elucidate different functions of the proteome.

Selective Enrichment of Monospecific Polyclonal Antibodies for Antibody-based Proteomics Efforts

Journal of Chromatography. A. Jul, 2004  |  Pubmed ID: 15317410

A high stringency protocol, suitable for systematic purification of polyclonal antibodies, is described. The procedure is designed to allow the generation of target protein-specific antibodies suitable for functional annotation of proteins. Antibodies were generated by immunization with recombinantly produced affinity-tagged target proteins. To obtain stringent recovery of the antibodies, a two-step affinity chromatography principle was devised to first deplete the affinity tag-specific antibodies followed by a second step for affinity capture of the target protein-specific antibodies. An analytical dot-blot array system was developed to analyze the cross-reactivity of the affinity-purified antibodies. The results suggest that the protocol can be used in a highly parallel and automated manner to generate mono-specific polyclonal antibodies for large-scale, antibody-based proteomics efforts, i.e. affinity proteomics.

Effect of Substrate Feed Rate on Recombinant Protein Secretion, Degradation and Inclusion Body Formation in Escherichia Coli

Applied Microbiology and Biotechnology. Jul, 2005  |  Pubmed ID: 15655679

The effect of changes in substrate feed rate during fedbatch cultivation was investigated with respect to soluble protein formation and transport of product to the periplasm in Escherichia coli. Production was transcribed from the P(malK) promoter; and the cytoplasmic part of the production was compared with production from the P(lacUV5) promoter. The fusion protein product, Zb-MalE, was at all times accumulated in the soluble protein fraction except during high-feed-rate production in the cytoplasm. This was due to a substantial degree of proteolysis in all production systems, as shown by the degradation pattern of the product. The product was also further subjected to inclusion body formation. Production in the periplasm resulted in accumulation of the full-length protein; and this production system led to a cellular physiology where the stringent response could be avoided. Furthermore, the secretion could be used to abort the diauxic growth phase resulting from use of the P(malK) promoter. At high feed rate, the accumulation of acetic acid, due to overflow metabolism, could furthermore be completely avoided.

Affinity Ligands for Industrial Protein Purification

Protein and Peptide Letters. May, 2005  |  Pubmed ID: 15907172

Significant efforts are put into the design of large-scale purification processes of proteins due to great demands regarding cost efficiency and safety. In order to design an effective purification scheme the unit operations need to be reduced to a minimum. In this review we are discussing proteinaceous ligands as well as small synthetic mimics for use in affinity chromatography for large-scale applications. Different advantages as well as drawbacks of the two approaches are outlined.

A Novel Flow Cytometry-based Method for Analysis of Expression Levels in Escherichia Coli, Giving Information About Precipitated and Soluble Protein

Journal of Biotechnology. Sep, 2005  |  Pubmed ID: 15996784

A high throughput method for screening of protein expression is described. By using a flow cytometer, levels of both soluble and precipitated protein can simultaneously be assessed in vivo. Protein fragments were fused to the N-terminus of enhanced GFP and the cell samples were analysed using a flow cytometer. Data concerning whole cell fluorescence and light scattering was collected. The whole cell fluorescence is probing intracellular concentrations of soluble fusion proteins. Concurrently, forward scattered light gives data about inclusion body formation, valuable information in process optimisation. To evaluate the method, the cells were disrupted, separated into soluble and non-soluble fractions and analysed by gel electrophoresis. A clear correlation between fluorescence and soluble target protein was shown. Interestingly, the distribution of the cells regarding forward scatter (standard deviation) correlates with the amount of inclusion bodies formed. Finally, the newly developed method was used to evaluate two different purification tags, His(6) and Z(basic), and their effect on the expression pattern.

High-throughput Solubility Assay for Purified Recombinant Protein Immunogens

Biochimica Et Biophysica Acta. Aug, 2005  |  Pubmed ID: 16084133

A high-throughput assay is described for analysis of the solubility of purified recombinant proteins. The assay is based on affinity purification of proteins in the presence of chaotropic agents followed by a dilution and incubation step to investigate the solubility in the absence of high concentrations of such agents. The assay can be performed in a 96-well format, which makes it well suited for high-throughput applications. For 125 recombinant proteins expressed as part of an antibody-based proteomics effort, experimental solubility data were compared to calculated hydrophobicity values based on the amino acid sequence of each protein. This comparison showed only weak correlation between the theoretical and experimental values, which emphasizes the importance of experimental assays to determine the solubility of recombinant proteins.

A Human Protein Atlas for Normal and Cancer Tissues Based on Antibody Proteomics

Molecular & Cellular Proteomics : MCP. Dec, 2005  |  Pubmed ID: 16127175

Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, approximately 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.

Towards a Human Proteome Atlas: High-throughput Generation of Mono-specific Antibodies for Tissue Profiling

Proteomics. Nov, 2005  |  Pubmed ID: 16237735

A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.

High-throughput Protein Purification Using an Automated Set-up for High-yield Affinity Chromatography

Protein Expression and Purification. Apr, 2006  |  Pubmed ID: 16483795

One of the key steps in high-throughput protein production is protein purification. A newly developed high-yield protein purification and isolation method for laboratory scale use is presented. This procedure allows fully automated purification of up to 60 cell lysates with milligram yields of pure recombinant protein in 18.5h. The method is based on affinity chromatography and has been set up on an instrument that utilizes positive pressure for liquid transfer through columns. A protocol is presented that includes all steps of equilibration of the chromatography resin, load of sample, wash, and elution without any manual handling steps. In contrast to most existing high-throughput protein purification procedures, positive pressure is used for liquid transfer rather than vacuum. Positive pressure and individual pumps for each liquid channel contribute to controlled flow rates and eliminate the risk of introducing air in the chromatography resin and therefore ensure stable chromatography conditions. The procedure is highly reproducible and allows for high protein yield and purity.

From Gene Expression Analysis to Tissue Microarrays: a Rational Approach to Identify Therapeutic and Diagnostic Targets in Lymphoid Malignancies

Molecular & Cellular Proteomics : MCP. Jun, 2006  |  Pubmed ID: 16524965

Mantle cell lymphoma (MCL) is an aggressive lymphoid malignancy for which better treatment strategies are needed. To identify potential diagnostic and therapeutic targets, a signature consisting of MCL-associated genes was selected based on a comprehensive gene expression analysis of malignant and normal B cells. The corresponding protein epitope signature tags were identified and used to raise monospecific, polyclonal antibodies, which were subsequently analyzed on paraffin-embedded sections of malignant and normal tissue. In this study, we demonstrate that the initial selection strategy of MCL-associated genes successfully allows identification of protein antigens either uniquely expressed or overexpressed in MCL compared with normal lymphoid tissues. We propose that genome-based, affinity proteomics, using protein epitope signature tag-induced antibodies, is an efficient way to rapidly identify a number of disease-associated protein candidates of both previously known and unknown identities.

Multiplexed PrEST Immunization for High-throughput Affinity Proteomics

Journal of Immunological Methods. Aug, 2006  |  Pubmed ID: 16949094

Monospecific antibodies dfdfdfdf (msAbs) generated through antigen specific purification of polyclonal antisera are valuable tools in proteome analyses. However, proteome wide generation of msAbs would require extensive immunization programs. Therefore, it would be desirable to develop efficient immunization and purification methods to reduce the number of animals needed for such antibody-based research. Here we describe a multiplex immunization strategy for generation of msAbs towards recombinantly produced human protein fragments, denoted PrESTs. Antisera from rabbits immunized with a mixture of two, three, five and up to ten different PrESTs have been purified by a two-step immunoaffinity-based protocol and the efficiency of the purification method was analyzed using a two-color protein array concept. The obtained results showed that almost 80% of the animals immunized with antigens composed of two or three different PrESTs yielded antibodies recognizing all the included PrESTs. Furthermore, the modified two-step purification method effectively eliminated all background binding and produced pure antibody pools against individual PrESTs. This indicates that the multiplexed PrEST immunization strategy described here could become useful for high-throughput antibody-based proteomics initiatives, thus significantly reducing the number of animals needed in addition to providing a more cost-efficient method for production of msAbs.

A Human Protein Atlas Based on Antibody Proteomics

Current Opinion in Molecular Therapeutics. Jun, 2006  |  Pubmed ID: 16774037

The Human Protein Atlas is a comprehensive database that provides the protein expression profiles for a large number of human proteins, presented as immunohistological images from most human tissues. This review provides an overview of the contents of the atlas, discusses the project strategy and highlights the importance of open access for data validation and quality. Essential procedures that are implemented during antibody production and image generation, such as the use of protein epitope signature tags (PrEST) antigens, monospecific antibodies, tissue microarrays and thorough quality validation, are also discussed. The Human Protein Atlas is related to four other expression atlas initiatives, including, in particular, an upcoming protein atlas developed by the Sanger Institute.

Microfluidic Analysis of Antibody Specificity in a Compact Disk Format

Journal of Proteome Research. Jul, 2006  |  Pubmed ID: 16823963

A new and flexible technology for high throughput analysis of antibody specificity and affinity is presented. The method is based on microfluidics and takes advantage of compact disks (CDs) in which the centrifugal force moves fluids through microstructures containing immobilized metal affinity chromatography columns. Analyses are performed as a sandwich assay, where antigen is captured to the column via a genetically attached His6-tag. The antibodies to be analyzed are applied onto the columns. Thereafter, fluorescently labeled secondary antibodies recognize the bound primary antibodies, and detection is carried out by laser-induced fluorescence. The CDs contain 104 microstructures enabling analysis of antibodies against more than 100 different proteins using a single CD. Importantly, through the three-dimensional visualization of the binding patterns in a column it is possible to separate high affinity from low affinity binding. The method presented here is shown to be very sensitive, flexible and reproducible.

Single-step Recovery and Solid-phase Refolding of Inclusion Body Proteins Using a Polycationic Purification Tag

Biotechnology Journal. Feb, 2006  |  Pubmed ID: 16892247

A strategy for purification of inclusion body-forming proteins is described, in which the positively charged domain Z(basic) is used as a fusion partner for capture of denatured proteins on a cation exchange column. It is shown that the purification tag is selective under denaturing conditions. Furthermore, the new strategy for purification of proteins from inclusion bodies is compared with the commonly used method for purification of His(6)-tagged inclusion body proteins. Finally, the simple and effective means of target protein capture provided by the Z(basic) tag is further successfully explored for solid-phase refolding. This procedure has the inherited advantage of combining purification and refolding in one step and offers the advantage of eluting the concentrated product in a suitable buffer.

Pyrosequencing: History, Biochemistry and Future

Clinica Chimica Acta; International Journal of Clinical Chemistry. Jan, 2006  |  Pubmed ID: 16165119

Pyrosequencing is a DNA sequencing technology based on the sequencing-by-synthesis principle.

The RBCC Gene RFP2 (Leu5) Encodes a Novel Transmembrane E3 Ubiquitin Ligase Involved in ERAD

Molecular Biology of the Cell. May, 2007  |  Pubmed ID: 17314412

RFP2, a gene frequently lost in various malignancies, encodes a protein with RING finger, B-box, and coiled-coil domains that belongs to the RBCC/TRIM family of proteins. Here we demonstrate that Rfp2 is an unstable protein with auto-polyubiquitination activity in vivo and in vitro, implying that Rfp2 acts as a RING E3 ubiquitin ligase. Consequently, Rfp2 ubiquitin ligase activity is dependent on an intact RING domain, as RING deficient mutants fail to drive polyubiquitination in vitro and are stabilized in vivo. Immunopurification and tandem mass spectrometry enabled the identification of several putative Rfp2 interacting proteins localized to the endoplasmic reticulum (ER), including valosin-containing protein (VCP), a protein indispensable for ER-associated degradation (ERAD). Importantly, we also show that Rfp2 regulates the degradation of the known ER proteolytic substrate CD3-delta, but not the N-end rule substrate Ub-R-YFP (yellow fluorescent protein), establishing Rfp2 as a novel E3 ligase involved in ERAD. Finally, we show that Rfp2 contains a C-terminal transmembrane domain indispensable for its localization to the ER and that Rfp2 colocalizes with several ER-resident proteins as analyzed by high-resolution immunostaining. In summary, these data are all consistent with a function for Rfp2 as an ERAD E3 ubiquitin ligase.

Approaches for Systematic Proteome Exploration

Biomolecular Engineering. Jun, 2007  |  Pubmed ID: 17376740

With the completion of the human genome project (HUGO) during recent years, gene function, protein abundance and expression patterns in tissues and cell types have emerged as central areas for the scientific community. A mapped human proteome will extend the value of the genome sequence and large-scale efforts aiming at elucidating protein localization, abundance and function are invaluable for biomarker and drug discovery. This research area, termed proteomics, is more demanding than any genome sequencing effort and to perform this on a wide scale is a highly diverse task. Therefore, the proteomics field employs a range of methods to examine different aspects of proteomics including protein localization, protein-protein interactions, posttranslational modifications and alteration of protein composition (e.g. differential expression) in tissues and body fluids. Here, some of the most commonly used methods, including chromatographic separations together with mass spectrometry and a number of affinity proteomics concepts are discussed and exemplified.

High-throughput Protein Purification Under Denaturating Conditions by the Use of Cation Exchange Chromatography

Biotechnology Journal. Jun, 2007  |  Pubmed ID: 17492715

A high-throughput protein purification strategy using the polycationic Z(basic) tag has been developed. In order for the strategy to be useful both for soluble and less soluble proteins, a denaturating agent, urea, was used in all purification steps. First, four target proteins were genetically fused to the purification tag, Z(basic). These protein constructs were purified by cation exchange chromatography and eluted using a salt gradient. From the data achieved, a purification strategy was planned including stepwise elution to enable parallel protein purification using a laboratory robot. A protocol that includes all steps, equilibration of the chromatography resin, load of sample, wash, and elution, all without any manual handling steps, was handled by the laboratory robot. The program allows automated purification giving milligram amounts of pure recombinant protein of up to 60 cell lysates. In this study 22 different protein constructs, with different characteristics regarding pI and solubility, were successfully purified by the laboratory robot. The data show that Z(basic) can be used as a general purification tag also under denaturating conditions. Moreover, the strategy enables purification of proteins with different pI and solubility using ion exchange chromatography (IEXC). The procedure is highly reproducible and allows for high protein yield and purity and is therefore a good complement to the commonly used His(6)-tag.

Z(basic)--a Novel Purification Tag for Efficient Protein Recovery

Journal of Chromatography. A. Aug, 2007  |  Pubmed ID: 17570380

A positively charged protein domain, Z(basic), can be used as a general purification tag to achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography. To construct a protein domain usable for ion-exchange chromatography, the surface of protein Z was engineered to be highly charged, which allowed for selective capture of target proteins on a cation-exchanger at physiological pH values. Interestingly, the novel domain, denoted Z(basic), was shown to be selective also under denaturing conditions and could preferably be used for purification of proteins solubilised from inclusion bodies. Moreover, a flexible process for solid-phase refolding was developed, using Z(basic) as a reversible linker to the cation-exchanger resin. This procedure has the inherited advantage of combining purification and refolding into a single step and still enabling elution of a concentrated product in a suitable buffer. This article summarizes development and use of the Z(basic) tag in small and pilot-plant-scale downstream processing.

Affibody-mediated Transferrin Depletion for Proteomics Applications

Biotechnology Journal. Nov, 2007  |  Pubmed ID: 17639529

An Affibody (Affibody) ligand with specific binding to human transferrin was selected by phage display technology from a combinatorial protein library based on the staphylococcal protein A (SpA)-derived Z domain. Strong and selective binding of the selected Affibody ligand to transferrin was demonstrated using biosensor technology and dot blot analysis. Impressive specificity was demonstrated as transferrin was the only protein recovered by affinity chromatography from human plasma. Efficient Affibody-mediated capture of transferrin, combined with IgG- and HSA-depletion, was demonstrated for human plasma and cerebrospinal fluid (CSF). For plasma, 85% of the total transferrin content in the samples was depleted after only two cycles of transferrin removal, and for CSF, 78% efficiency was obtained in single-step depletion. These results clearly suggest a potential for the development of Affibody-based resins for the removal of abundant proteins in proteomics analyses.

A Web-based Tool for in Silico Biomarker Discovery Based on Tissue-specific Protein Profiles in Normal and Cancer Tissues

Molecular & Cellular Proteomics : MCP. May, 2008  |  Pubmed ID: 17913849

Here we report the development of a publicly available Web-based analysis tool for exploring proteins expressed in a tissue- or cancer-specific manner. The search queries are based on the human tissue profiles in normal and cancer cells in the Human Protein Atlas portal and rely on the individual annotation performed by pathologists of images representing immunohistochemically stained tissue sections. Approximately 1.8 million images representing more than 3000 antibodies directed toward human proteins were used in the study. The search tool allows for the systematic exploration of the protein atlas to discover potential protein biomarkers. Such biomarkers include tissue-specific markers, cell type-specific markers, tumor type-specific markers, markers of malignancy, and prognostic or predictive markers of cancers. Here we show examples of database queries to generate sets of candidate biomarker proteins for several of these different categories. Expression profiles of candidate proteins can then subsequently be validated by examination of the underlying high resolution images. The present study shows examples of search strategies revealing several potential protein biomarkers, including proteins specifically expressed in normal cells and in cancer cells from specified tumor types. The lists of candidate proteins can be used as a starting point for further validation in larger patient cohorts using both immunological approaches and technologies utilizing more classical proteomics tools.

MAP20, a Microtubule-associated Protein in the Secondary Cell Walls of Hybrid Aspen, is a Target of the Cellulose Synthesis Inhibitor 2,6-dichlorobenzonitrile

Plant Physiology. Nov, 2008  |  Pubmed ID: 18805954

We have identified a gene, denoted PttMAP20, which is strongly up-regulated during secondary cell wall synthesis and tightly coregulated with the secondary wall-associated CESA genes in hybrid aspen (Populus tremula x tremuloides). Immunolocalization studies with affinity-purified antibodies specific for PttMAP20 revealed that the protein is found in all cell types in developing xylem and that it is most abundant in cells forming secondary cell walls. This PttMAP20 protein sequence contains a highly conserved TPX2 domain first identified in a microtubule-associated protein (MAP) in Xenopus laevis. Overexpression of PttMAP20 in Arabidopsis (Arabidopsis thaliana) leads to helical twisting of epidermal cells, frequently associated with MAPs. In addition, a PttMAP20-yellow fluorescent protein fusion protein expressed in tobacco (Nicotiana tabacum) leaves localizes to microtubules in leaf epidermal pavement cells. Recombinant PttMAP20 expressed in Escherichia coli also binds specifically to in vitro-assembled, taxol-stabilized bovine microtubules. Finally, the herbicide 2,6-dichlorobenzonitrile, which inhibits cellulose synthesis in plants, was found to bind specifically to PttMAP20. Together with the known function of cortical microtubules in orienting cellulose microfibrils, these observations suggest that PttMAP20 has a role in cellulose biosynthesis.

A Genecentric Human Protein Atlas for Expression Profiles Based on Antibodies

Molecular & Cellular Proteomics : MCP. Oct, 2008  |  Pubmed ID: 18669619

An attractive path forward in proteomics is to experimentally annotate the human protein complement of the genome in a genecentric manner. Using antibodies, it might be possible to design protein-specific probes for a representative protein from every protein-coding gene and to subsequently use the antibodies for systematical analysis of cellular distribution and subcellular localization of proteins in normal and disease tissues. A new version (4.0) of the Human Protein Atlas has been developed in a genecentric manner with the inclusion of all human genes and splice variants predicted from genome efforts together with a visualization of each protein with characteristics such as predicted membrane regions, signal peptide, and protein domains and new plots showing the uniqueness (sequence similarity) of every fraction of each protein toward all other human proteins. The new version is based on tissue profiles generated from 6120 antibodies with more than five million immunohistochemistry-based images covering 5067 human genes, corresponding to approximately 25% of the human genome. Version 4.0 includes a putative list of members in various protein classes, both functional classes, such as kinases, transcription factors, G-protein-coupled receptors, etc., and project-related classes, such as candidate genes for cancer or cardiovascular diseases. The exact antigen sequence for the internally generated antibodies has also been released together with a visualization of the application-specific validation performed for each antibody, including a protein array assay, Western blot analysis, immunohistochemistry, and, for a large fraction, immunofluorescence-based confocal microscopy. New search functionalities have been added to allow complex queries regarding protein expression profiles, protein classes, and chromosome location. The new version of the protein atlas thus is a resource for many areas of biomedical research, including protein science and biomarker discovery.

Evaluation of Monospecific Antibodies: a Comparison Study with Commercial Analogs Using Immunohistochemistry on Tissue Microarrays

Applied Immunohistochemistry & Molecular Morphology : AIMM / Official Publication of the Society for Applied Immunohistochemistry. Oct, 2008  |  Pubmed ID: 18685494

Generation of monospecific antibodies (msAbs) (multiepitope) through affinity purification of polyclonal antisera is a plausible strategy for high-throughput production of affinity reagents toward large sets of proteins. These antibodies are generated using readily accessible gene sequence information from publicly available databases. The resulting antibodies have the potential to be used in a variety of assays, probing differentially presented and altered proteins with high sensitivity and specificity. In the present study, 48 msAbs were compared with corresponding commercial analogs. Immunohistochemical staining properties were evaluated on tissue microarrays, representing various normal human tissues from 144 different individuals. MsAbs showed similar immunostaining patterns as compared with corresponding commercial analogs in 44 out of totally 48 (92%) antibody pairs analyzed. Although only few antibody pairs showed major discrepancies, minor dissimilarities were frequently seen. Our results suggest that msAbs are reliable and valuable tools in antibody-based proteomics, enabling analysis of protein expression patterns in cells and tissues. High-throughput strategies employing such antibodies provide a consistent approach in the exploration of the human proteome.

High-throughput Protein Production--lessons from Scaling Up from 10 to 288 Recombinant Proteins Per Week

Biotechnology Journal. Jan, 2009  |  Pubmed ID: 19039781

The demand for high-throughput recombinant protein production has markedly increased with the increased activity in the field of proteomics. Within the Human Protein Atlas project recombinantly produced human protein fragments are used for antibody production. Here we describe how the protein expression and purification protocol has been optimized in the project to allow for handling of nearly 300 different proteins per week. The number of manual handling steps has been significantly reduced (from 18 to 9) and the protein purification has been completely automated.

Automated Sample Preparation Method for Mass Spectrometry Analysis on Recombinant Proteins

Journal of Chromatography. A. May, 2009  |  Pubmed ID: 19345368

A completely automated procedure for the purification and desalting of proteins with a polyhistidine purification tag prior to mass spectrometry analysis is presented. The system is ideal for rapid quality control and optimization studies and it provides researchers with a straightforward, reliable tool for studies of recombinant proteins. Forty-eight samples can be prepared within 4.5h and only small cultivation and buffer volumes are needed. In this proof of concept, 19,000-35,000Da recombinant proteins from both crude and clarified cell lysates were successfully prepared for subsequent analysis by electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry as well as by gel electrophoresis.

Tissue Profiling of the Mammalian Central Nervous System Using Human Antibody-based Proteomics

Molecular & Cellular Proteomics : MCP. Jul, 2009  |  Pubmed ID: 19351664

A need exists for mapping the protein profiles in the human brain both during normal and disease conditions. Here we studied 800 antibodies generated toward human proteins as part of a Human Protein Atlas program and investigated their suitability for detailed analysis of various levels of a rat brain using immuno-based methods. In this way, the parallel, rather limited analysis of the human brain, restricted to four brain areas (cerebellum, cerebral cortex, hippocampus, and lateral subventricular zone), could be extended in the rat model to 25 selected areas of the brain. Approximately 100 antibodies (12%) revealed a distinct staining pattern and passed validation of specificity using Western blot analysis. These antibodies were applied to coronal sections of the rat brain at 0.7-mm intervals covering the entire brain. We have now produced detailed protein distribution profiles for these antibodies and acquired over 640 images that form the basis of a publicly available portal of an antibody-based Rodent Brain Protein Atlas database (www.proteinatlas.org/rodentbrain). Because of the systematic selection of target genes, the majority of antibodies included in this database are generated against proteins that have not been studied in the brain before. Furthermore optimized tissue processing and colchicine treatment allow a high quality, more extended annotation and detailed analysis of subcellular distributions and protein dynamics.

Development of Affinity Columns for the Removal of High-abundance Proteins in Cerebrospinal Fluid

Biotechnology and Applied Biochemistry. Feb, 2009  |  Pubmed ID: 18412540

Various approaches for removal of high-abundance components in body fluids are currently available. While most methods are constructed for plasma depletion, there is a need for body-fluid-specific strategies. The aim of the present study was to design an affinity matrix suitable for the depletion of high-abundance proteins in CSF (cerebrospinal fluid). Hence, molecules with specific affinity towards proteins present at high concentration in CSF were desired. Affibody molecules are specific binders of small size that have shown high stability under various conditions and are therefore good candidates for such a matrix. The protein composition in CSF resembles that in plasma. However, 20% of the proteins are brain-derived and are therefore present in higher proportions in CSF than in plasma, whereas larger plasma-derived proteins are less abundant in CSF. Therefore five high-abundance CSF proteins were chosen for the design of a CSF-specific depletion setup. Affibody molecules with specificity towards HSA (human serum albumin), IgG, transferrin and transthyretin were combined in an affinity column. In addition, polyclonal antibodies against cystatin C were coupled to chromatographic beads and packed in a separate column. Highly reproducible and efficient removal of the five target proteins was observed. The proportion of depleted proteins were estimated to be 99, 95, 74, 92 and 83% for HSA, IgG, transferrin, transthyretin and cystatin C respectively. SDS/PAGE analysis was used for monitoring and identifying proteins in native CSF, depleted CSF samples and the captured fractions. Moreover, shotgun proteomics was used for protein identification in native as well as depleted CSF and the achieved data were compared. Enhanced identification of lower-abundance components was observed in the depleted fraction, in terms of more detected peptides per protein.

Generation and Validation of Affinity Reagents on a Proteome-wide Level

Journal of Molecular Recognition : JMR. Mar-Apr, 2009  |  Pubmed ID: 18546091

There is a need for protein-specific affinity reagents to explore the gene products encoded by the genome. Recently, systematic efforts to generate validated affinity reagents on a whole human proteome level have been initiated. There are several issues for such efforts, including choice of antigen, type of affinity reagent, and the subsequent validation of the generated protein-specific binders. The advantages and disadvantages with the different approaches are discussed and the problems related to quality assessment of antibodies to be used in multi-platform applications are addressed. This review also describes the efforts to create a virtual resource of validated antibodies using a community-based portal and summarizes the status and visions for the publicly available human protein atlas (http://www.proteinatlas.org) showing the human protein profiles in a large number of normal and cancer tissues as well as a large set of human cell lines.

Editorial: Biotech in the Post Genomic Era

Biotechnology Journal. Dec, 2009  |  Pubmed ID: 20014219

A Global View of Protein Expression in Human Cells, Tissues, and Organs

Molecular Systems Biology. 2009  |  Pubmed ID: 20029370

Defining the protein profiles of tissues and organs is critical to understanding the unique characteristics of the various cell types in the human body. In this study, we report on an anatomically comprehensive analysis of 4842 protein profiles in 48 human tissues and 45 human cell lines. A detailed analysis of over 2 million manually annotated, high-resolution, immunohistochemistry-based images showed a high fraction (>65%) of expressed proteins in most cells and tissues, with very few proteins (<2%) detected in any single cell type. Similarly, confocal microscopy in three human cell lines detected expression of more than 70% of the analyzed proteins. Despite this ubiquitous expression, hierarchical clustering analysis, based on global protein expression patterns, shows that the analyzed cells can be still subdivided into groups according to the current concepts of histology and cellular differentiation. This study suggests that tissue specificity is achieved by precise regulation of protein levels in space and time, and that different tissues in the body acquire their unique characteristics by controlling not which proteins are expressed but how much of each is produced.

Affibody Molecule-mediated Depletion of HSA and IgG Using Different Buffer Compositions: a 15 Min Protocol for Parallel Processing of 1-48 Samples

Biotechnology and Applied Biochemistry. Jun, 2010  |  Pubmed ID: 20446920

High-abundant plasma proteins pose a challenge in a large number of proteomics-based technologies. Depletion of these high-abundant proteins has proven to be a fruitful strategy to circumvent masking of lower-abundant proteins that could serve as valuable biomarker candidates. However, current strategies often do not meet the throughput requirements of large-scale proteomic studies. In the present paper, a flexible and parallelized method for the depletion of high-abundant proteins is described, allowing the removal of the two most abundant proteins from 48 blood-derived samples in less than 15 min using Affibody molecules as affinity ligands. A sample-processing platform like this should be suitable for a number of proteomics technologies; its flexibility in buffer composition allows for different types of downstream applications.

A Small Bispecific Protein Selected for Orthogonal Affinity Purification

Biotechnology Journal. Jun, 2010  |  Pubmed ID: 20518064

A novel protein domain with dual affinity has been created by randomization and selection. The small alkali-stabilized albumin-binding domain (ABD*), used as scaffold to construct the library, has affinity to human serum albumin (HSA) and is constituted of 46 amino acids of which 11 were randomized. To achieve a dual binder, the binding site of the inherent HSA affinity was untouched and the randomization was made on the opposite side of the molecule. Despite its small size and randomization of almost a quarter of its amino acids, a bifunctional molecule, ABDz1, with ability to bind to both HSA and the Z2 domain/protein A was successfully selected using phage display. Moreover, the newly selected variant showed improved affinity for HSA compared to the parental molecule. This novel protein domain has been characterized regarding secondary structure and affinity to the two different ligands. The possibility for affinity purification on two different matrices has been investigated using the two ligands, the HSA matrix and the protein A-based, MabSelect SuRe matrix, and the new protein domain was purified to homogeneity. Furthermore, gene fusions between the new domain and three different target proteins with different characteristics were made. To take advantage of both affinities, a purification strategy referred to as orthogonal affinity purification using two different matrices was created. Successful purification of all three versions was efficiently carried out using this strategy.

Towards a Knowledge-based Human Protein Atlas

Nature Biotechnology. Dec, 2010  |  Pubmed ID: 21139605

Editorial: Global Biotech Challenges

Biotechnology Journal. Dec, 2010  |  Pubmed ID: 21154666

The Impact of Tissue Fixatives on Morphology and Antibody-based Protein Profiling in Tissues and Cells

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society. Mar, 2010  |  Pubmed ID: 19901271

Pathology archives harbor large amounts of formalin-fixed, paraffin-embedded tissue samples, used mainly in clinical diagnostics but also for research purposes. Introduction of heat-induced antigen retrieval has enabled the use of tissue samples for extensive immunohistochemical analysis, despite the fact that antigen retrieval may not recover all epitopes, owing to alterations of the native protein structure induced by formalin. The aim of this study was to investigate how different fixatives influence protein recognition by immunodetection methods in tissues, cell preparations, and protein lysates, as compared with formalin. Seventy-two affinity-purified polyclonal antibodies were used to evaluate seven different fixatives. The aldehyde-based fixative Glyo-fixx proved to be excellent for preservation of proteins in tissue detected by immunohistochemistry (IHC), similar to formalin. A non-aldehyde-based fixative, NEO-FIX was superior for fixation of cultured cells, in regard to morphology, and thereby also advantageous for IHC. Large variability in the amount of protein extracted from the differently fixed tissues was observed, and the HOPE fixative provided the overall highest yield of protein. In conclusion, morphological resolution and immunoreactivity were superior in tissues fixed with aldehyde-based fixatives, whereas the use of non-aldehyde-based fixatives can be advantageous in obtaining high protein yield for Western blot analysis. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Targeted Protein Pullout from Human Tissue Samples Using Competitive Elution

Biotechnology Journal. Jan, 2011  |  Pubmed ID: 21170982

One commonly used strategy to gain information on the proteins in a cell is to isolate the proteins of interest by specific binders, often antibodies. Not only the specificity of the capturing antibodies but also the washing and elution conditions are crucial to avoid false-positive protein identifications. Eluting the target protein from the matrix, while avoiding the release of unrelated background proteins, should both provide more correct information on the target protein and its interaction partners, and minimize the effort to perform downstream analyses through the reduced number of eluted proteins. In this study, a novel approach for selective protein pullout is presented. Monospecific antibodies were used to selectively pullout target proteins from a complex biosample. Subsequently, the target proteins were competitively eluted from the affinity media with the recombinant antigen. To deplete the antigen from the eluted sample, IMAC spin columns were utilized to bind the N-terminal His-tag of the antigens. The competitive elution method was applied both to a model system, and for the extraction of a native human target protein. In the model system the recombinant target protein BBC7 was spiked into a protein extract of human liver, whereas an endogenously expressed target protein, cTAGE5, was extracted from the liver extract directly. SDS-PAGE analysis and mass spectrometry confirmed affinity isolation of expected target proteins. More selective elution was obtained using the competitive procedure as compared to elution at low pH. Competitive elution has thus been shown to offer an effective approach for wide-scale pullout experiments where proteins and their interaction partners are to be studied.

Enhancing the Protein Production Levels in Escherichia Coli with a Strong Promoter

The FEBS Journal. Mar, 2011  |  Pubmed ID: 21205203

In biotechnology, the use of Escherichia coli for recombinant protein production has a long tradition, although the optimal production conditions for certain proteins are still not evident. The most favorable conditions for protein production vary with the gene product. Temperature and induction conditions represent parameters that affect total protein production, as well as the amount of soluble protein. Furthermore, the choice of promoter and bacterial strain will have large effects on the production of the target protein. In the present study, the effects of three different promoters (T7, trc and lacUV5) on E. coli production of target proteins with different characteristics are presented. The total amount of target protein as well as the amount of soluble protein were analyzed, demonstrating the benefits of using a strong promoter such as T7. To understand the underlying causes, transcription levels have been correlated with the total amount of target protein and protein solubility in vitro has been correlated with the amount of soluble protein that is produced. In addition, the effects of two different E. coli strains, BL21(DE3) and Rosetta(DE3), on the expression pattern were analyzed. It is concluded that the regulation of protein production is a combination of the transcription and translation efficiencies. Other important parameters include the nucleotide-sequence itself and the solubility of the target protein.

Surrogate Antigens As Targets for Proteome-wide Binder Selection

New Biotechnology. Jul, 2011  |  Pubmed ID: 21232647

In the past decade, many initiatives were taken for the development of antibodies for proteome-wide studies, as well as characterisation and validation of clinically relevant disease biomarkers. Phage display offers many advantages compared to antibody generation by immunisation because it is an unlimited resource of affinity reagents without batch-to-batch variation and is also amendable for high throughput in contrast to conventional hybridoma technology. One of the major bottlenecks to proteome-wide binder selection is the limited supply of suitable target antigens representative of the human proteome. Here, we provide proof of principle of using easily accessible, cancer-associated protein epitope signature tags (PrESTs), routinely generated within the Human Protein Atlas project, as surrogate antigens for full-length proteins in phage selections for the retrieval of target-specific binders. These binders were subsequently tested in western blot, immunohistochemistry and protein microarray application to demonstrate their functionality.

Parallel Production and Verification of Protein Products Using a Novel High-throughput Screening Method

Biotechnology Journal. Aug, 2011  |  Pubmed ID: 21681961

Protein production and analysis in a parallel fashion is today applied in laboratories worldwide and there is a great need to improve the techniques and systems used for this purpose. In order to save time and money, a fast and reliable screening method for analysis of protein production and also verification of the protein product is desired. Here, a micro-scale protocol for the parallel production and screening of 96 proteins in plate format is described. Protein capture was achieved using immobilized metal affinity chromatography and the product was verified using matrix-assisted laser desorption ionization time-of-flight MS. In order to obtain sufficiently high cell densities and product yield in the small-volume cultivations, the EnBase® cultivation technology was applied, which enables cultivation in as small volumes as 150 μL. Here, the efficiency of the method is demonstrated by producing 96 human, recombinant proteins, both in micro-scale and using a standard full-scale protocol and comparing the results in regard to both protein identity and sample purity. The results obtained are highly comparable to those acquired through employing standard full-scale purification protocols, thus validating this method as a successful initial screening step before protein production at a larger scale.

Translational Database Selection and Multiplexed Sequence Capture for Up Front Filtering of Reliable Breast Cancer Biomarker Candidates

PloS One. 2011  |  Pubmed ID: 21698250

Biomarker identification is of utmost importance for the development of novel diagnostics and therapeutics. Here we make use of a translational database selection strategy, utilizing data from the Human Protein Atlas (HPA) on differentially expressed protein patterns in healthy and breast cancer tissues as a means to filter out potential biomarkers for underlying genetic causatives of the disease. DNA was isolated from ten breast cancer biopsies, and the protein coding and flanking non-coding genomic regions corresponding to the selected proteins were extracted in a multiplexed format from the samples using a single DNA sequence capture array. Deep sequencing revealed an even enrichment of the multiplexed samples and a great variation of genetic alterations in the tumors of the sampled individuals. Benefiting from the upstream filtering method, the final set of biomarker candidates could be completely verified through bidirectional Sanger sequencing, revealing a 40 percent false positive rate despite high read coverage. Of the variants encountered in translated regions, nine novel non-synonymous variations were identified and verified, two of which were present in more than one of the ten tumor samples.

Editorial: Biotech Reviews - Keeping Up with Current Developments

Biotechnology Journal. Sep, 2011  |  Pubmed ID: 21910253

High Nuclear RBM3 Expression is Associated with an Improved Prognosis in Colorectal Cancer

Proteomics. Clinical Applications. Dec, 2011  |  Pubmed ID: 21956899

In this study, we investigated the prognostic impact of human RBM3 expression in colorectal cancer using tissue microarray-based immunohistochemical analysis.

Engineering Bispecificity into a Single Albumin-binding Domain

PloS One. 2011  |  Pubmed ID: 21991353

Bispecific antibodies as well as non-immunoglobulin based bispecific affinity proteins are considered to have a very high potential in future biotherapeutic applications. In this study, we report on a novel approach for generation of extremely small bispecific proteins comprised of only a single structural domain. Binding to tumor necrosis factor-α (TNF-α) was engineered into an albumin-binding domain while still retaining the original affinity for albumin, resulting in a bispecific protein composed of merely 46 amino acids. By diversification of the non albumin-binding side of the three-helix bundle domain, followed by display of the resulting library on phage particles, bispecific single-domain proteins were isolated using selections with TNF-α as target. Moreover, based on the obtained sequences from the phage selection, a second-generation library was designed in order to further increase the affinity of the bispecific candidates. Staphylococcal surface display was employed for the affinity maturation, enabling efficient isolation of improved binders as well as multiparameter-based sortings with both TNF-α and albumin as targets in the same selection cycle. Isolated variants were sequenced and the binding to albumin and TNF-α was analyzed. This analysis revealed an affinity for TNF-α below 5 nM for the strongest binders. From the multiparameter sorting that simultaneously targeted TNF-α and albumin, several bispecific candidates were isolated with high affinity to both antigens, suggesting that cell display in combination with fluorescence activated cell sorting is a suitable technology for engineering of bispecificity. To our knowledge, the new binders represent the smallest engineered bispecific proteins reported so far. Possibilities and challenges as well as potential future applications of this novel strategy are discussed.

Covalent Immunoglobulin Labeling Through a Photoactivable Synthetic Z Domain

Bioconjugate Chemistry. Dec, 2011  |  Pubmed ID: 22026370

Traditionally, labeling of antibodies has been performed by covalent conjugation to amine or carboxyl groups. These methods are efficient but suffer from nonspecificity, since all free and available amine/carboxyl groups have the possibility to react. This drawback may lead to uncontrolled levels and locations of the labeling. Hence, the labeled molecules might behave differently and, possibly, the binding site of the antibody will also be affected. In this project, we have developed a highly stringent method for labeling of antibodies by utilizing an immunoglobulin-binding domain from protein A, the Z domain. Domain Z has been synthesized with an amino acid analogue, benzoylphenylalanine, capable of forming covalent attachment to other amino acids upon UV-exposure. This feature has been used for directed labeling of immunoglobulins and subsequent use of these in different assays.