Directed evolution of single-chain Fv for cytoplasmic expression using the β-galactosidase complementation assay results in proteins highly susceptible to protease degradation and aggregation

BACKGROUND: Antibody fragments are molecules widely used for diagnosis and therapy. A large amount of protein is frequently required for such applications. New approaches using folding reporter enzymes have recently been proposed to increase soluble expression of foreign proteins in Escherichia coli. To date, these methods have only been used to screen for proteins with better folding properties but have never been used to select from a large library of mutants. In this paper we apply one of these methods to select mutations that increase the soluble expression of two antibody fragments in the cytoplasm of E. coli. RESULTS: We used the β-galactosidase α-complementation system to monitor and evolve two antibody fragments for high expression levels in E. coli cytoplasm. After four rounds of mutagenesis and selection from large library repertoires (>10(7 )clones), clones exhibiting high levels of β-galactosidase activity were isolated. These clones expressed a higher amount of soluble fusion protein than the wild type in the cytoplasm, particularly in a strain deficient in the cytoplasmic Lon protease. The increase in the soluble expression level of the unfused scFv was, however, much less pronounced, and the unfused proteins proved to be more aggregation prone than the wild type. In addition, the soluble expression levels were not correlated with the β-galactosidase activity present in the cells. CONCLUSION: This is the first report of a selection for soluble protein expression using a fusion reporter method. Contrary to anticipated results, high enzymatic activity did not correlate with the soluble protein expression level. This was presumably due to free α-peptide released from the protein fusion by the host proteases. This means that the α-complementation assay does not sense the fusion expression level, as hypothesized, but rather the amount of free released α-peptide. Thus, the system does not select, in our case, for higher soluble protein expression level but rather for higher protease susceptibility of the fusion protein

Hematopoietic-Prostaglandin D2 synthase through PGD2 production is involved in the adult ovarian physiology

<p>Abstract</p> <p>Background</p> <p>The prostaglandin D2 (PGD2) pathway is involved in numerous biological processes and while it has been identified as a partner of the embryonic sex determining male cascade, the roles it plays in ovarian function remain largely unknown. PGD2 is secreted by two prostaglandin D synthases (Pgds); the male-specific lipocalin (L)-Pgds and the hematopoietic (H)-Pgds.</p> <p>Methods</p> <p>To study the expression of the Pgds in the adult ovary, <it>in situ </it>hybridization were performed. Then, to evaluate the role of H-Pgds produced PGD2 in the ovarian physiology, adult female mice were treated with HQL-79, a specific inhibitor of H-Pgds enzymatic activity. The effects on expression of the gonadotrophin receptors <it>FshR </it>and <it>LhR</it>, steroidogenic genes <it>Cyp11A1</it>, <it>StAR </it>and on circulating progesterone and estradiol, were observed.</p> <p>Results</p> <p>We report the localization of <it>H-Pgds </it>mRNA in the granulosa cells from the primary to pre-ovulatory follicles. We provide evidence of the role of H-Pgds-produced PGD2 signaling in the FSH signaling through increased <it>FshR </it>and <it>LhR </it>receptor expression. This leads to the activation of steroidogenic <it>Cyp11A1 </it>and <it>StAR </it>gene expression leading to progesterone secretion, independently on other prostanoid-synthetizing mechanisms. We also identify a role whereby H-Pgds-produced PGD2 is involved in the regulation of follicular growth through inhibition of granulosa cell proliferation in the growing follicles.</p> <p>Conclusions</p> <p>Together, these results show PGD2 signaling to interfere with FSH action within granulosa cells, thus identifying an important and unappreciated role for PGD2 signaling in modulating the balance of proliferation, differentiation and steroidogenic activity of granulosa cells.</p

Is hypospadias a genetic, endocrine or environmental disease, or still an unexplained malformation?

Q1Artículo de revisión187-197Hypospadias is one of the most frequent genital malformations in the male newborn and results from an abnormal penile and urethral development. This process requires a correct genetic programme, time- and space-adapted cellular differentiation, complex tissue interactions, and hormonal mediation through enzymatic activities and hormonal transduction signals. Any disturbance in these regulations may induce a defect in the virilization of the external genitalia and hypospadias. This malformation thus appears to be at the crossroads of various mechanisms implicating genetic and environmental factors. The genes of penile development (HOX, FGF, Shh) and testicular determination (WT1, SRY) and those regulating the synthesis [luteinizing hormone (LH) receptor] and action of androgen (5a reductase, androgen receptor) can cause hypospadias if altered. Several chromosomal abnormalities and malformative syndromes include hypospadias, from anterior to penoscrotal forms. More recently, CXorf6 and ATF3 have been reported to be involved. Besides these genomic and hormonal factors, multiple substances found in the environment can also potentially interfere with male genital development because of their similarity to hormones. The proportion of hypospadias cases for which an aetiology is detected varies with the authors but it nevertheless remains low, especially for less severe cases. An interaction between genetic background and environment is likely

Using Experimental Models to Decipher the Effects of Acetaminophen and NSAIDs on Reproductive Development and Health

Nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin (acetylsalicylic acid), diclofenac and ibuprofen (IBU), and analgesic drugs, such as acetaminophen (APAP, or paracetamol), are widely used to treat inflammation and pain. APAP and IBU are over-the-counter drugs and are among the most commonly taken drugs in the first trimester of pregnancy, even in combination. Furthermore, these drugs and their metabolites are released in the environment, and can be frequently detected in wastewater, surface water, and importantly in drinking water. Although their environmental concentrations are much lower than the therapeutics doses, this suggests an uncontrolled low-dose exposure of the general population, including pregnant women and young children, two particularly at risk populations. Epidemiological studies show that exposure to these molecules in the first and second trimester of gestation can favor genital malformations in new-born boys. To investigate the cellular, molecular and mechanistic effects of exposure to these molecules, ex vivo studies with human or rodent gonadal explants and in vivo experiments in rodents have been performed in the past years. This review recapitulates recent data obtained in rodent models after in utero or postnatal exposure to these drugs. The first part of this review discusses the mechanisms by which NSAIDs and analgesics may impair gonadal development and maturation, puberty development, sex hormone production, maturation and function of adult organs, and ultimately fertility in the exposed animals and their offspring. Like other endocrine disruptors, NSAIDs and APAP interfere with endocrine gland function and may have inter/transgenerational adverse effects. Particularly, they may target germ cells, resulting in reduced quality of male and female gametes, and decreased fertility of exposed individuals and their descendants. Then, this review discusses the effects of exposure to a single drug (APAP, aspirin, or IBU) or to combinations of drugs during early embryogenesis, and the consequences on postnatal gonadal development and adult reproductive health. Altogether, these data may increase medical and public awareness about these reproductive health concerns, particularly in women of childbearing age, pregnant women, and parents of young children

A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm

<p>Abstract</p> <p>Background</p> <p>Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus.</p> <p>Results</p> <p>We describe the construction and validation of a large synthetic human single chain antibody fragment library based on a unique framework and optimized for cytoplasmic expression. Focusing the library by mimicking the natural diversity of CDR3 loops ensured that the scFvs were fully human and functional. We show that the library is highly diverse and functional since it has been possible to isolate by phage-display several strong binders against the five proteins tested in this study, the Syk and Aurora-A protein kinases, the αβ tubulin dimer, the papillomavirus E6 protein and the core histones. Some of the selected scFvs are expressed at an exceptional high level in the bacterial cytoplasm, allowing the purification of 1 mg of active scFv from only 20 ml of culture. Finally, we show that after three rounds of selection against core histones, more than half of the selected scFvs were active when expressed <it>in vivo </it>in human cells since they were essentially localized in the nucleus.</p> <p>Conclusion</p> <p>This new library is a promising tool not only for an easy and large-scale selection of functional intrabodies but also for the isolation of highly expressed scFvs that could be used in numerous biotechnological and therapeutic applications.</p

Screening of MAMLD1 Mutations in 70 Children with 46,XY DSD: Identification and Functional Analysis of Two New Mutations

More than 50% of children with severe 46,XY disorders of sex development (DSD) do not have a definitive etiological diagnosis. Besides gonadal dysgenesis, defects in androgen biosynthesis, and abnormalities in androgen sensitivity, the Mastermind-like domain containing 1 (MAMLD1) gene, which was identified as critical for the development of male genitalia, may be implicated. The present study investigated whether MAMLD1 is implicated in cases of severe 46,XY DSD and whether routine sequencing of MAMLD1 should be performed in these patients

Clinical, Biological and Genetic Analysis of Prepubertal Isolated Ovarian Cyst in 11 Girls

BACKGROUND: The cause of isolated gonadotropin-independent precocious puberty (PP) with an ovarian cyst is unknown in the majority of cases. Here, we describe 11 new cases of peripheral PP and, based on phenotypes observed in mouse models, we tested the hypothesis that mutations in the GNAS1, NR5A1, LHCGR, FSHR, NR5A1, StAR, DMRT4 and NOBOX may be associated with this phenotype. METHODOLOGY/PRINCIPAL FINDINGS: 11 girls with gonadotropin-independent PP were included in this study. Three girls were seen for a history of prenatal ovarian cyst, 6 girls for breast development, and 2 girls for vaginal bleeding. With one exception, all girls were seen before 8 years of age. In 8 cases, an ovarian cyst was detected, and in one case, suspected. One other case has polycystic ovaries, and the remaining case was referred for vaginal bleeding. Four patients had a familial history of ovarian anomalies and/or infertility. Mutations in the coding sequences of the candidate genes GNAS1, NR5A1, LHCGR, FSHR, NR5A1, StAR, DMRT4 and NOBOX were not observed. CONCLUSIONS/SIGNIFICANCE: Ovarian PP shows markedly different clinical features from central PP. Our data suggest that mutations in the GNAS1, NR5A1, LHCGR, FSHR StAR, DMRT4 and NOBOX genes are not responsible for ovarian PP. Further research, including the identification of familial cases, is needed to understand the etiology of ovarian PP

Construction et optimisation d anticorps intracellulaires pour l analyse in vivo des protéines

L immunisation intracellulaire utilise des fragments d anticorps (intracorps) exprimés à l intérieur de la cellule afin de se lier à une protéine et d inhiber sa fonction. Le problème majeur limitant l utilisation des intracorps est l absence de formation des ponts disulfures en milieu cytoplasmique (réducteur). En utilisant un fragment d anticorps, le scFv 13R4, sélectionné pour sa forte expression et sa très bonne solubilité dans le cytoplasme malgré l absence des ponts disulfures, nous avons construit par greffage des boucles hypervariables (CDR) un anticorps dirigé contre l'oncoprotéine E6 du papillomavirus. L'anticorps obtenu après optimisation présente des caractéristiques d'expression et de solubilité proche de l'anticorps 13R4 originel en ayant acquis les propriétés de reconnaissance de l'anticorps anti-protéine E6.Toutefois, devant les difficultés rencontrées pour obtenir des intracorps par greffages des boucles hypervariables, nous avons créé une banque d'anticorps optimisés pour l'immunisation intracellulaire. Pour cela, différentes boucles hypervariables ont été greffées sur la charpente de l'anticorps 13R4 en respectant la diversité des CDR observée dans les anticorps naturels humains. Cette banque a été testée contre différentes cibles intracellulaires et confirme son utilité pour isoler rapidement des intracorps.Intracellular immunization consists in the expression of antibody fragments inside the cell that bind to a protein and interfer with its function. The main problem limiting the use of intrabodies is the absence of disulfide bonds in the reducing conditions pertaining in the cell cytoplasm. We used the scFv13R4 antibody fragment, selected for its high soluble expression level in the cytoplasm, to construct, by loop (CDR) grafting, an antibody fragment against the E6 protein of human papillomavirus type 16. After several optimization steps, cytoplasmic soluble expression of the grafted anti-E6-protein antibody fragment was comparable to that of the original antibody (13R4).To circumvent this long procedure, we designed and constructed an optimised library of antibody fragments for intracellular immunization, based on the scFv13R4 framework with randomized CDR3 loops mimicking the natural diversity of human CDR loop sequences. This library has been tested against several proteins, demonstrating that it is now possible to rapidly obtain intrabodies against any protein.MONTPELLIER-BU Pharmacie (341722105) / SudocSudocFranceF

Immunisation intracellulaire et problèmes d'expression intracytoplasmique (construction d'un Intracorps anti-protéine E6 du Papillomavirus)

MONTPELLIER-BU Pharmacie (341722105) / SudocSudocFranceF

SDS-PAGE of soluble and insoluble extracts (5 μl) of the best clones isolated after four rounds of mutagenesis, cloned in the pET23NN plasmid

<p><b>Copyright information:</b></p><p>Taken from "Directed evolution of single-chain Fv for cytoplasmic expression using the β-galactosidase complementation assay results in proteins highly susceptible to protease degradation and aggregation"</p><p>Microbial Cell Factories 2004;3():16-16.</p><p>Published online 17 Dec 2004</p><p>PMCID:PMC544847.</p><p>Copyright © 2004 Philibert and Martineau; licensee BioMed Central Ltd.</p> Proteins were revealed either by Coomassie staining (top) or by western blot using the 9E10 anti-c-myc monoclonal antibody followed by an anti-mouse alkaline phosphatase-conjugated antibody and detected with the chromogenic substrate BCIP/NBT. A) HuLys11 scFv and mutants; B) 225.28S scFv and mutants