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1.  The Availability of a Recombinant Anti-SNAP Antibody in VHH Format Amplifies the Application Flexibility of SNAP-Tagged Proteins 
Antibodies are indispensable reagents in basic research, and those raised against tags constitute a useful tool for the evaluation of the biochemistry and biology of novel proteins. In this paper, we describe the isolation and characterization of a single-domain recombinant antibody (VHH) specific for the SNAP-tag, using Twist2 as a test-protein. The antibody was efficient in western blot, immunoprecipitation, immunopurification, and immunofluorescence. The sequence corresponding to the anti-SNAP has been subcloned for large-scale expression in vectors that allow its fusion to either a 6xHis-tag or the Fc domain of rabbit IgG2 taking advantage of a new plasmid that was specifically designed for VHH antibodies. The two different fusion antibodies were compared in immunopurification and immunofluorescence experiments, and the recombinant protein SNAP-Twist2 was accurately identified by the anti-SNAP Fc-VHH construct in the nuclear/nucleolar subcellular compartment. Furthermore, such localization was confirmed by direct Twist2 identification by means of anti-Twisit2 VHH antibodies recovered after panning of the same naïve phage display library used to isolate the anti-SNAP binders. Our successful localization of Twist2 protein using the SNAP-tag-based approach and the anti-Twist2-specific recombinant single-domain antibodies opens new research possibilities in this field.
PMCID: PMC2963136  PMID: 20981296
2.  Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies 
The isolation of recombinant antibody fragments from displayed libraries represents a powerful alternative to the generation of IgGs using hybridoma technology. The selected antibody fragments can then be easily engineered into (multi)-tagged constructs of variable mass and complexity as well as reconstituted into Camelidae IgG-like molecules when expressed fused to Fc domains. Nevertheless, all antibody constructs depend on an oxidizing environment for correct folding and consequently still belong to the proteins difficult to express in bacteria. In such organisms they are mostly produced at low yields in the periplasmic space.
We demonstrate that fusion constructs of recombinant antibodies in combination with multiple tags can be produced at high yields and totally functional in the cytoplasm of bacteria expressing sulfhydryl oxidase. The method was applied to structurally demanding molecules such as VHHs fused to SNAP and Fc domains and was validated using the antibody-derived reagents in a variety of immune techniques (FACS, ELISA, WB, IP, SPR, and IF).
The collected data demonstrate the feasibility of a method that establishes a totally new approach for producing rapidly and inexpensively functional Camelidae IgG-like monoclonal antibodies and antibody-based reagents containing multiple disulfide bonds and suitable for both basic research and clinical applications.
Electronic supplementary material
The online version of this article (doi:10.1186/s12934-014-0140-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4172947  PMID: 25223348
Affinity purification; Avidity effect; Disulfide bonds; Immune diagnostics; Single-chain antibodies; Single-domain antibodies; Sortase; Sulfhydryl oxidase
3.  The Trip Adviser guide to the protein science world: a proposal to improve the awareness concerning the quality of recombinant proteins 
BMC Research Notes  2014;7:585.
In many research articles, where protein purification is required for various assays, (protein-protein interactions, activity assays, etc.), we always have access to the final results, but seldom have access to the raw data required for an accurate evaluation of the protein quality. This data is extremely important on one hand to critically evaluate the quality of the proteins used in the described research and, on the other hand, to allow other laboratories to safely use the described procedure in a reproducible manner. We herby propose to include a standardized methodology that can easily be incorporated in research papers. Moreover, this methodology can be utilized as a “quality control” ladder, where the more information given, will lead to a higher ranking of the article. This “quality control” stamp will allow researchers retrieving relevant and useful materials and methods in the field of protein research.
Electronic supplementary material
The online version of this article (doi:10.1186/1756-0500-7-585) contains supplementary material, which is available to authorized users.
PMCID: PMC4161829  PMID: 25178166
Data annotation; Data reproducibility; Protein quality
4.  Recombinant polypeptide production in E. coli: towards a rational approach to improve the yields of functional proteins 
The development of complementary technologies enabled the successful production of recombinant polypeptides in bacteria and opened to biology researchers new avenues as obtaining suitable amounts of proteins necessary for their experimental work became easy, fast, and inexpensive. Nevertheless, the recombinant approach remained somehow unpredictable, since many constructs resisted to apparent production or accumulated as aggregates.
Several factors and physical/chemical conditions that could improve the accumulation of native-like protein were identified. At the same time, it was acknowledged that the outcome of most of them was erratic and that almost any protein required its own specific optimized set of conditions to achieve its correct folding. The attempt to understand the critical points specific for recombinant protein production missed the goal of setting universally useful protocols, but contributed to the increase of the rate of success by proposing always new empiric combinations.
Nevertheless, the results published in the recent literature allow for a better comprehension of some key mechanisms controlling protein production in E. coli and could enable the elaboration of rational methodologies for improving the quantitative and qualitative features of the produced polypeptides. This result will be achieved when the identification of the limiting step that impairs the accomplishment of the native folding for any single construct will become straightforward. This minireview will discuss how factors such as the expression rate, the folding machinery, and the secretion efficiency may impact the final protein yields.
PMCID: PMC3832250  PMID: 24176192
Carrier protein; Chaperones; Expression rate; Fusion proteins; Inclusion bodies; Isomerases; Promoter; Regulative sequences; Secretion mechanisms
5.  Recent contributions in the field of the recombinant expression of disulfide bonded proteins in bacteria 
The production of heterologous disulfide bonded proteins in bacteria remains a biotechnological challenge. A rapid literature survey results in the identification of some interesting proposals, such as the option of producing functional proteins in the cytoplasm in the presence of sulfhydryl oxidases and isomerases. Furthermore, an ever-increasing number of applications refers to recombinant proteins displayed at the bacterial surface. Time will tell whether these developments will lead to universally accepted laboratory protocols.
PMCID: PMC3462667  PMID: 22978724
7.  Biotechnological applications of recombinant single-domain antibody fragments 
Single-domain antibody fragments possess structural features, such as a small dimension, an elevated stability, and the singularity of recognizing epitopes non-accessible for conventional antibodies that make them interesting for several research and biotechnological applications.
The discovery of the single-domain antibody's potentials has stimulated their use in an increasing variety of fields. The rapid accumulation of articles describing new applications and further developments of established approaches has made it, therefore, necessary to update the previous reviews with a new and more complete summary of the topic.
Beside the necessary task of updating, this work analyses in detail some applicative aspects of the single-domain antibodies that have been overseen in the past, such as their efficacy in affinity chromatography, as co-crystallization chaperones, protein aggregation controllers, enzyme activity tuners, and the specificities of the unconventional single-domain fragments.
PMCID: PMC3123181  PMID: 21658216
8.  Meeting Report from the Second “Minimum Information for Biological and Biomedical Investigations” (MIBBI) workshop 
Standards in Genomic Sciences  2010;3(3):259-266.
This report summarizes the proceedings of the second workshop of the ‘Minimum Information for Biological and Biomedical Investigations’ (MIBBI) consortium held on Dec 1-2, 2010 in Rüdesheim, Germany through the sponsorship of the Beilstein-Institute. MIBBI is an umbrella organization uniting communities developing Minimum Information (MI) checklists to standardize the description of data sets, the workflows by which they were generated and the scientific context for the work. This workshop brought together representatives of more than twenty communities to present the status of their MI checklists and plans for future development. Shared challenges and solutions were identified and the role of MIBBI in MI checklist development was discussed. The meeting featured some thirty presentations, wide-ranging discussions and breakout groups. The top outcomes of the two-day workshop as defined by the participants were: 1) the chance to share best practices and to identify areas of synergy; 2) defining a series of tasks for updating the MIBBI Portal; 3) reemphasizing the need to maintain independent MI checklists for various communities while leveraging common terms and workflow elements contained in multiple checklists; and 4) revision of the concept of the MIBBI Foundry to focus on the creation of a core set of MIBBI modules intended for reuse by individual MI checklist projects while maintaining the integrity of each MI project. Further information about MIBBI and its range of activities can be found at
PMCID: PMC3035314  PMID: 21304730
9.  The binding of NCAM to FGFR1 induces a specific cellular response mediated by receptor trafficking 
The Journal of Cell Biology  2009;187(7):1101-1116.
Although FGF-2 causes the FGFR to be internalized and degraded, NCAM gets cells moving by stabilizing the receptor, promoting receptor recycling, and initiating a promigratory signaling cascade.
Neural cell adhesion molecule (NCAM) associates with fibroblast growth factor (FGF) receptor-1 (FGFR1). However, the biological significance of this interaction remains largely elusive. In this study, we show that NCAM induces a specific, FGFR1-mediated cellular response that is remarkably different from that elicited by FGF-2. In contrast to FGF-induced degradation of endocytic FGFR1, NCAM promotes the stabilization of the receptor, which is recycled to the cell surface in a Rab11- and Src-dependent manner. In turn, FGFR1 recycling is required for NCAM-induced sustained activation of various effectors. Furthermore, NCAM, but not FGF-2, promotes cell migration, and this response depends on FGFR1 recycling and sustained Src activation. Our results implicate NCAM as a nonconventional ligand for FGFR1 that exerts a peculiar control on the intracellular trafficking of the receptor, resulting in a specific cellular response. Besides introducing a further level of complexity in the regulation of FGFR1 function, our findings highlight the link of FGFR recycling with sustained signaling and cell migration and the critical role of these events in dictating the cellular response evoked by receptor activation.
PMCID: PMC2806277  PMID: 20038681
10.  Monodispersity of recombinant Cre recombinase correlates with its effectiveness in vivo 
BMC Biotechnology  2009;9:80.
Cre recombinase is a common reagent used for the in vivo on/off switching of the expression of target genes flanked by loxP sites. In particular, recombinant TAT-Cre fusion constructs purified from bacteria have been used to promote the cell uptake of the enzyme. However, the recovery of active TAT-Cre remains a demanding process and its specific activity varies significantly among batches, making difficult data comparison.
We noticed a strong correlation between recombinase activity and enzyme monodispersity. The existence of such correlation enabled us to indirectly monitor the TAT-Cre recombinase activity during the multi-step purification process by measuring its monodispersity, a parameter detectable by means of a spectrofluorimetric assay that allows the calculation of the Aggregation Index (AI) in an easy and rapid way. AI values were recorded after each purification passage to identify the critical steps and to choose optimal alternatives for chromatographic conditions, desalting procedures, and protocols for bacterial endotoxin removal. Furthermore, the effect of metal ions and temperature on TAT-Cre aggregation and inactivation was characterized in vitro. Finally, we optimized the enzyme delivery protocol in vivo by following the accumulation tuning of the reporter protein β-catenin.
A rational purification protocol for TAT-Cre has been developed by choosing the options that minimize the enzyme aggregation. Our data suggest that AI measurement should support the optimization of any protocol aiming at the recovery of monodispersed protein.
PMCID: PMC2755479  PMID: 19747375
11.  Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli 
Bacteria are simple and cost effective hosts for producing recombinant proteins. However, their physiological features may limit their use for obtaining in native form proteins of some specific structural classes, such as for instance polypeptides that undergo extensive post-translational modifications. To some extent, also the production of proteins that depending on disulfide bridges for their stability has been considered difficult in E. coli.
Both eukaryotic and prokaryotic organisms keep their cytoplasm reduced and, consequently, disulfide bond formation is impaired in this subcellular compartment. Disulfide bridges can stabilize protein structure and are often present in high abundance in secreted proteins. In eukaryotic cells such bonds are formed in the oxidizing environment of endoplasmic reticulum during the export process. Bacteria do not possess a similar specialized subcellular compartment, but they have both export systems and enzymatic activities aimed at the formation and at the quality control of disulfide bonds in the oxidizing periplasm.
This article reviews the available strategies for exploiting the physiological mechanisms of bactera to produce properly folded disulfide-bonded proteins.
PMCID: PMC2689190  PMID: 19442264
12.  Minimal information: an urgent need to assess the functional reliability of recombinant proteins used in biological experiments 
Structural characterization of proteins used in biological experiments is largely neglected. In most publications, the information available is totally insufficient to judge the functionality of the proteins used and, therefore, the significance of identified protein-protein interactions (was the interaction specific or due to unspecific binding of misfolded protein regions?) or reliability of kinetic and thermodynamic data (how much protein was in its native form?). As a consequence, the results of single experiments might not only become questionable, but the whole reliability of systems biology, built on these fundaments, would be weakened.
The introduction of Minimal Information concerning purified proteins to add as metadata to the main body of a manuscript would render straightforward the assessment of their functional and structural qualities and, consequently, of results obtained using these proteins. Furthermore, accepted standards for protein annotation would simplify data comparison and exchange. This article has been envisaged as a proposal for aggregating scientists who share the opinion that the scientific community needs a platform for Minimum Information for Protein Functionality Evaluation (MIPFE).
PMCID: PMC2499994  PMID: 18647423
13.  Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli 
BMC Biotechnology  2007;7:32.
The overproduction of recombinant proteins in host cells often leads to their misfolding and aggregation. Previous attempts to increase the solubility of recombinant proteins by co-overproduction of individual chaperones were only partially successful. We now assessed the effects of combined overproduction of the functionally cooperating chaperone network of the E. coli cytosol on the solubility of recombinant proteins.
A two-step procedure was found to show the strongest enhancement of solubility. In a first step, the four chaperone systems GroEL/GroES, DnaK/DnaJ/GrpE, ClpB and the small HSPs IbpA/IbpB, were coordinately co-overproduced with recombinant proteins to optimize de novo folding. In a second step, protein biosynthesis was inhibited to permit chaperone mediated refolding of misfolded and aggregated proteins in vivo. This novel strategy increased the solubility of 70% of 64 different heterologous proteins tested up to 42-fold.
The engineered E. coli strains and the two-step procedure presented here led to a remarkable increase in the solubility of a various recombinant proteins and should be applicable to a wide range of target proteins produced in biotechnology.
PMCID: PMC1904446  PMID: 17565681
14.  Heating as a rapid purification method for recovering correctly-folded thermotolerant VH and VHH domains 
BMC Biotechnology  2007;7:7.
Recombinant antibodies from Camelidae (VHHs) are potentially useful tools for both basic research and biotechnological applications because of their small size, robustness, easy handling and possibility to refold after chemio-physical denaturation. Their heat tolerance is a particularly interesting feature because it has been recently related to both high yields during recombinant expression and selective purification of folded protein.
Purification of recombinant RE3 VHH by heat treatment yielded the same amount of antibody as purification by affinity chromatography and negligible differences were found in stability, secondary structure and functionality. Similar results were obtained using another class of thermotolerant proteins, the single domain VH scaffold, described by Jespers et al. [8]. However, thermosensitive VHs could not withstand the heat treatment and co-precipitated with the bacterial proteins. In both cases, the thermotolerant proteins unfolded during the treatment but promptly refolded when moved back to a compatible temperature.
Heat treatment can simplify the purification protocol of thermotolerant proteins as well as remove any soluble aggregate. Since the re-folding capability after heat-induced denaturation was previously correlated to higher performance during recombinant expression, a unique heating step can be envisaged to screen constructs that can provide high yields of correctly-folded proteins.
PMCID: PMC1790891  PMID: 17257422
15.  The evaluation of the factors that cause aggregation during recombinant expression in E. coli is simplified by the employment of an aggregation-sensitive reporter 
The yields of soluble recombinant proteins expressed in bacteria are often low due to the tendency of the heterologous proteins to form aggregates. Therefore, aggregation reporters have been envisaged to simplify the comparison among different expression conditions and to speed up the identification of suitable protocols that improve the solubility. The probe we used is composed by an IbpAB promoter specifically activated by protein aggregates fused to a sequence coding the β-galactosidase, the activity of which becomes, therefore, indicative of the aggregation degree.
The collected data show that the probe is reliable in terms of reproducibility inside a range of experimental conditions and faster and more sensitive than the analysis methods based on SDS-PAGE and successive western blot.
The β-galactosidase probe was useful to identify which parameters could influence the aggregation of the model proteins and to set up an optimized expression protocol. The effect of growth temperature, induction modality, co-expression with molecular chaperones and addition of osmolytes on the accumulation of aggregates were evaluated following the β-galactosidase activity. Interestingly, a significant correlation was observed between estimated decreased aggregation and higher yields of soluble protein.
We also compared a set of expression vectors with various regulative features and found that the single characteristics, like promoter, copy number or polymerase, were not relevant for controlling the recombinant protein aggregation whilst the crucial factor resulted being the total expression rate of the system.
The aggregation reporter used in our experiments represents a useful tool to evaluate the different factors that can be modulated to optimize a recombinant expression protocol. Furthermore, the rapid estimation of the aggregation degree enables to discriminate this from other causes responsible for scarce recombinant yields.
PMCID: PMC1564408  PMID: 16948849
16.  Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol–overexpressed molecular chaperones 
Cell Stress & Chaperones  2005;10(4):329-339.
When massively expressed in bacteria, recombinant proteins often tend to misfold and accumulate as soluble and insoluble nonfunctional aggregates. A general strategy to improve the native folding of recombinant proteins is to increase the cellular concentration of viscous organic compounds, termed osmolytes, or of molecular chaperones that can prevent aggregation and can actively scavenge and convert aggregates into natively refoldable species. In this study, metal affinity purification (immobilized metal ion affinity chromatography [IMAC]), confirmed by resistance to trypsin digestion, was used to distinguish soluble aggregates from soluble nativelike proteins. Salt-induced accumulation of osmolytes during induced protein synthesis significantly improved IMAC yields of folding-recalcitrant proteins. Yet, the highest yields were obtained with cells coexpressing plasmid-encoded molecular chaperones DnaK-DnaJ-GrpE, ClpB, GroEL-GroES, and IbpA/B. Addition of the membrane fluidizer heat shock–inducer benzyl alcohol (BA) to the bacterial medium resulted in similar high yields as with plasmid-mediated chaperone coexpression. Our results suggest that simple BA-mediated induction of endogenous chaperones can substitute for the more demanding approach of chaperone coexpression. Combined strategies of osmolyte-induced native folding with heat-, BA-, or plasmid-induced chaperone coexpression can be thought to optimize yields of natively folded recombinant proteins in bacteria, for research and biotechnological purposes.
PMCID: PMC1283876  PMID: 16333986
17.  Simplified screening for the detection of soluble fusion constructs expressed in E. coli using a modular set of vectors 
The solubility of recombinant proteins expressed in bacteria is often disappointingly low. Several strategies have been developed to improve the yield and one of the most common strategies is the fusion of the target protein with a suitable partner. Despite several reports on the successful use of each of these carriers to increase the solubility of some recombinant proteins, none of them was always successful and a combinatorial approach seems more efficient to identify the optimal combination for a specific protein. Therefore, the efficiency of an expression system critically depends on the speed in the identification of the optimal combination for the suitable fusion candidate in a screening process. This paper describes a set of expression vectors (pETM) designed for rapid subcloning, expression and subsequent purification using immobilized metal affinity chromatography (IMAC).
A single PCR product of two Yellow Fluorescent Proteins (EYFPs) was cloned into 18 vectors comprising identical restriction sites and varying fusion partners as well as differing protease recognition sites. After a small-scale expression, the yields of the different constructs were compared using a Coomassie stained SDS-polyacrylamide gel and the results of this preliminary screening were then confirmed by large-scale purification. The yields were calculated and the stability of the different constructs determined using three independent conditions. The results indicated a significant correlation between the length and composition of non-native amino acid tails and stability. Furthermore, the buffer specificity of TEV and 3C proteases was tested using fusion proteins differing only in their protease recognition sequence, and a His-GST-EYFP construct was employed to compare the efficiency of the two alternative affinity purification methods.
The experiments showed that the set of pETM vectors could be used for the rapid production of a large array of different constructs with specific yield, stability, and cleavage features. Their comparison allowed the identification of the optimal constructs to use for the large-scale expression. We expect that the approach outlined in this paper, i.e. the possibility to obtain in parallel fusion products of the target protein with different partners for a preliminary evaluation, would be highly beneficial for all them who are interested in the rapid identification of the optimal conditions for protein expression.
PMCID: PMC1326211  PMID: 16351710
18.  Characterization of the aggregates formed during recombinant protein expression in bacteria 
BMC Biochemistry  2005;6:10.
The first aim of the work was to analyze in detail the complexity of the aggregates formed upon overexpression of recombinant proteins in E. coli. A sucrose step gradient succeeded in separating aggregate subclasses of a GFP-GST fusion protein with specific biochemical and biophysical features, providing a novel approach for studying recombinant protein aggregates.
The total lysate separated into 4 different fractions whereas only the one with the lowest density was detected when the supernatant recovered after ultracentrifugation was loaded onto the sucrose gradient. The three further aggregate sub-classes were otherwise indistinctly precipitated in the pellet. The distribution of the recombinant protein among the four subclasses was strongly dependent on the DnaK availability, with larger aggregates formed in Dnak- mutants. The aggregation state of the GFP-GST recovered from each of the four fractions was further characterized by examining three independent biochemical parameters. All of them showed an increased complexity of the recombinant protein aggregates starting from the top of the sucrose gradient (lower mass aggregates) to the bottom (larger mass aggregates). These results were also confirmed by electron microscopy analysis of the macro-structure formed by the different aggregates. Large fibrils were rapidly assembled when the recombinant protein was incubated in the presence of cellular extracts, but the GFP-GST fusion purified soon after lysis failed to undergo amyloidation, indicating that other cell components probably participate in the active formation of large aggregates. Finally, we showed that aggregates of lower complexity are more efficiently disaggregated by a combination of molecular chaperones.
An additional analytical tool is now available to investigate the aggregation process and separate subclasses by their mass. It was possible to demonstrate the complexity of the aggregation pattern of a recombinant protein expressed in bacteria and to characterize biochemically the different aggregate subclasses. Furthermore, we have obtained evidence that the cellular environment plays a role in the development of the aggregates and the problem of the artifact generation of aggregates has been discussed using in vitro models. Finally, the possibility of separating aggregate fractions with different complexities offers new options for biotechnological strategies aimed at improving the yield of folded and active recombinant proteins.
PMCID: PMC1175841  PMID: 15927061
19.  A step ahead: combining protein purification and correct folding selection 
The success of recombinant protein expression seems unpredictable and even good yields of soluble proteins do not guarantee the correct folding. The search for soluble constructs can be performed by exploiting libraries and speeded up by automation, but these approaches are money and time consuming and the tags used for affinity purification can mask the real stability of the target proteins. The ideal purification protocol would include the structure quality control. A recent paper commented in this article describes a phage-display method to screen for antibodies that are able to re-fold after heat-denaturation and can be selectively affinity-purified only if monodispersed. It turned out that the proteins with high recovery performance after heat-shock were also suitable for efficient recombinant expression.
PMCID: PMC524515  PMID: 15471550
20.  The Vaccinia Virus E8R Gene Product: a Viral Membrane Protein That Is Made Early in Infection and Packaged into the Virions' Core 
Journal of Virology  2002;76(19):9773-9786.
Vaccinia virus (VV), a member of the poxvirus family, is unique among most other DNA viruses in that both transcription and DNA replication occur in the cytoplasm of the host cell. It was recently shown by electron microscopy (EM) that soon after viral DNA synthesis is initiated in HeLa cells, the replication sites become enwrapped by the membrane of the endoplasmic reticulum (ER). In the same study, a novel VV membrane protein, the E8R gene product, that may play a role in the ER wrapping process was identified (N. Tolonen, L. Doglio, S. Schleich, and J. Krijnse Locker, Mol. Biol. Cell 12:2031-2046, 2001). In the present study, the gene product of E8R was characterized both biochemically and morphologically. We show that E8R is made predominantly early in infection but is packaged into the virion. On two-dimensional gel electrophoresis, the protein appeared as a single spot throughout the VV life cycle; however, in the assembled virion, the protein underwent several modifications which resulted in a change in its molecular weight and its isoelectric point. EM of labeled cryosections of infected HeLa cells showed that the protein localized to the ER and to membranes located on one side of the Golgi complex as early as 1 h postinfection. Late in infection, E8R was additionally associated with membranes of immature virions and with intracellular mature viruses. Although E8R is predominantly associated with membranes, we show that the protein is associated with viral cores; the protein is present in cores made with NP-40-dithiothreitol as well as in incoming cores, the result of the viral entry process, early in infection. Finally, we show that E8R can be phosphorylated in vitro by the viral kinase F10L. It is able to bind DNA in vitro, and this binding may be modulated by phosphorylation by F10L. A putative role of the E8R gene product throughout the VV life cycle is discussed.
PMCID: PMC136492  PMID: 12208956

Results 1-20 (20)