Recombinant human TIM-3 ectodomain expressed in bacteria and recovered from inclusion bodies as a stable and active molecule.

Introduction: Microbial systems, such as Escherichia coli, as host recombinant expression is the most versatile and the cheapest system for protein production, however, several obstacles still remain, such as recovery of soluble and functional proteins from inclusion bodies, elimination of lipopolysaccharides (LPS) contamination, incomplete synthesis, degradation by proteases, and the lack of post-translational modifications, which becomes even more complex when comes to membrane proteins, because they are difficult not only to produce but also to keep in solution in its active state. T-cell Immunoglobulin and Mucin domain 3 (TIM-3) is a type I transmembrane protein that is predominantly expressed on the surface of T lymphocytes, natural killer (NK) cells, dendritic cells, and macrophages, playing a role as a negative immune checkpoint receptor. TIM-3 comprises a single ectodomain for interaction with immune system soluble and cellular components, a transmembrane domain, and a cytoplasmic tail, responsible for the binding of signaling and scaffolding molecules. TIM-3 pathway holds potential as a therapeutic target for immunotherapy against tumors, autoimmunity, chronic virus infections, and various malignancies, however, many aspects of the biology of this receptor are still incompletely understood, especially regarding its ligands. Methods: Here we overcome, for the first time, the challenge of the production of active immune checkpoint protein recovered from bacterial cytoplasmic inclusion bodies, being able to obtain an active, and non-glycosylated TIM-3 ectodomain (TIM-3-ECD), which can be used as a tool to better understand the interactions and roles of this immune checkpoint. The TIM-3 refolding was obtained by the association of high pressure and alkaline pH. Results: The purified TIM-3-ECD showed the correct secondary structure and was recognized from anti-TIM-3 structural-dependent antibodies likewise commercial TIM-3-ECD was produced by a mammal cells system. Furthermore, immunofluorescence showed the ability of TIM-3-ECD to bind to the surface of lung cancer A549 cells and to provide an additional boost for the expression of the lymphocyte activation marker CD69 in anti-CD3/CD28 activated human PBMC. Discussion: Taken together these results validated a methodology able to obtain active checkpoint proteins from bacterial inclusion bodies, which will be helpful to further investigate the interactions of this and others not yet explored immune checkpoints.

[Antimicrobial susceptibility of Gram-negative bacilli of community acquired intra-abdominal infections in a hospital at Buenos Aires, Argentina]. / Sensiblidad antimicrobiana de bacilos gramnegativos de infecciones intraabdominales de la comunidad en un hospital de la ciudad de Buenos Aires, Argentina.

OBJECTIVE: Community acquired complicated intra-abdominal infections (cIAI) are a common condition. Few data are available about the level of antimicrobial resistance of Gram-negative bacteria isolated from community acquired cIAIs in Argentina. METHODS: Retrospective-prospective observational study (March 2010 to February 2012). Gram-negative bacteria antimicrobial susceptibility of isolates from community acquired cIAIs were evaluated. RESULTS: During this period, a total of 85 patients were included and 138 pathogens were collected. Male sex: 58%. Median age: 33. Monomicrobial cultures were obtained in 49% of the cases. Ninety (65%) corresponded to Gram-negative organisms, and 48 (38%) to Gram-positive cocci. Gram-negative organisms most frequently observed were: Escherichia coli 76%, Klebsiella pneumoniae 8%, Pseudomonas aeruginosa 7% and Enterobacter spp. 6%. E. coli and K. pneumoniae showed a high percentage of strains resistance to ciprofloxacin of 37% and 29%, respectively. Similarly, resistance to ampicillin/sulbactam was observed in a 16% of the E. coli isolates. The prevalence of multiresistant Gram-negative organisms was 38%. CONCLUSIONS: A high level of resistance to antimicrobials was observed in community acquired cIAIs, mainly to ciprofloxacin and ampicillin/sulbactam two of the most used antimicrobial for empirically treatment of cIAIs in our country. In addition a significant proportion of multiresistant Gram-negative organisms were identified.

Acute necrotizing pancreatitis: can tigecycline be included in a therapeutic strategy?

INTRODUCTION: Acute necrotizing pancreatitis is a severe and life-threatening disease. Infection, which occurs in about 30% of cases, is the most feared complication. Antibiotic therapy is still discussed and there are no clear recommendation in literature. These clinical series underline the importance of having a clear antibiotic protocol, including tigecycline, in the management of acute necrotizing pancreatitis. Clinical series. Six patients with clinical and radiological diagnosis of necrotizing acute pancreatitis are treated in Emergency Surgery Department, following a conservative management, which includes fluid resuscitation, intensive care unit and radiological monitoring, ultrasound-guided percutaneous drainage and an antibiotic treatment protocol, that includes tigecycline. No one of the six patient undergo surgery (mean hospital stay: 44 days). In a six months follow-up all patients are alive and in good clinical conditions. DISCUSSION: Infection is the most important factor which determinate prognosis and outcome of acute necrotizing pancreatitis. Antibiotic prophylaxis is still discussed and there are no clear antibiotic treatment guidelines in literature. Despite its side effects on pancreatic gland, tigecycline is successful in resolution of sepsis, caused by infected pancreatic necrosis. CONCLUSIONS: Collaboration with infectivologist and a clear antibiotic protocol is fundamental to solve infected necrosis. Antibiotic treatment, set up as soon as possible, is successful in our six patients, as they recover without undergoing surgical procedures. Tigecycline offers broad coverage and efficacy against resistant pathogens for the treatment of documented pancreatic necrosis infection. However, further studies are necessary to fully understand the safety profile and efficacy of tigecycline.

Improving the therapeutic potential of endostatin by fusing it with the BAX BH3 death domain.

Endostatin (ES) inhibits angiogenesis, reducing tumor growth in animal models. However, it has low therapeutic effect in human clinical trials. BAX is a member of the BCL-2 family of proteins; its proapoptotic (BH3) domain interacts with other members of the family in the cytoplasm, to induce apoptosis. Here, we fused the BAX BH3 domain with murine ES, to enhance ES potency. Endothelial cells specifically internalize the fusion protein ES-BAX. The presence of the BAX domain enhances endothelial cell death by apoptosis by 1.8-fold and diminishes microvessel outgrowth in the rat aortic ring assay by 6.5-fold. Daily injections of 15 µg of ES-BAX/g in tumor-bearing mice reduce tumor weight by 86.9% as compared with ES-treated animals. Co-immunoprecipitation assays confirmed that ES-BAX interacts with members of the BCL-2 family. Also, ES interacts with BCL-2, BCL-XL, and BAK in endothelial cell lysates, suggesting a potential new mechanism for the apoptosis induction by ES. The superiority of the ES-BAX antiangiogenic effect indicates that this fusion protein could be a promising therapeutic alternative to treat cancer.

Effect of pressure on refolding of recombinant pentameric cholera toxin B.

The production of recombinant proteins is an essential tool for the expansion of modern biological research and biotechnology. The expression of heterologous proteins in Escherichia coli often results in an incomplete folding process that leads to the accumulation of inclusion bodies (IB), aggregates that hold a certain degree of native-like secondary structure. High hydrostatic pressure (HHP) impairs intermolecular hydrophobic and electrostatic interactions, leading to dissociation of aggregates under non-denaturing conditions and is therefore a useful tool to solubilize proteins for posterior refolding. Cholera toxin (CT) is composed of a non-toxic pentamer of B subunits (CTB), a useful adjuvant in vaccines, and a toxic subunit A (CTA). We studied the process of refolding of CTB using HHP. HHP was shown to be effective for dissociation of CTB monomers from IB. Posterior incubation at atmospheric pressure of concentrated CTB (1mg/ml) is necessary for the association of the monomers. Pentameric CTB was obtained when suspensions of CTB IB were compressed at 2.4kbar for 16h in the presence of Tween 20 and incubated at 1bar for 120h. Soluble and biologically active pentameric CTB was obtained, with a yield of 213mg CTB/liter of culture. The experience gained in this study can be important to improve the refolding of proteins with quaternary structure.

Continuous and high-level in vivo delivery of endostatin from recombinant cells encapsulated in TheraCyte immunoisolation devices.

Endostatin (ES) is a potent inhibitor of angiogenesis and tumor growth. Continuous ES delivery of ES improves the efficacy and potency of the antitumoral therapy. The TheraCyte system is a polytetrafluoroethylene (PTFE) semipermeable membrane macroencapsulation system for implantation of genetically engineered cells specially designed for the in vivo delivery of therapeutic proteins, such as ES, which circumvents the problem of limited half-life and variation in circulating levels. In order to enable neovascularization at the tissues adjacent to the devices prior to ES secretion by the cells inside them, we designed a scheme in which empty TheraCyte devices were preimplanted SC into immunodeficient mice. Only after healing (17 days later) were Chinese hamster ovary cells expressing ES injected into the preimplanted devices. In another model for device implantation, the cells expressing ES where loaded into the immunoisolation devices prior to implantation into the animals, and the TheraCyte were then immediately implanted SC into the mice. Throughout the 2-month study, constant high ES levels of up to 3.7 microg/ml were detected in the plasma of the mice preimplanted with the devices, while lower but also constant levels of ES (up to 2.1 microg/ml plasma) were detected in the mice that had received devices preloaded with the ES-expressing cells. Immunohistochemistry using anti-ES antibody showed reaction within the device and outside it, demonstrating that ES, secreted by the confined recombinant cells, permeated through the membrane and reached the surrounding tissues.

Cloning of serine protease cDNAs from Crotalus durissus terrificus venom gland and expression of a functional Gyroxin homologue in COS-7 cells.

Gyroxin is one of main serine proteases of Crotalus durissus terrificus venom, representing about 2% of the protein content in the crude venom. It is a 33 kDa glycoprotein with 3.8% by weight of sugar moiety. This toxin induces hemotoxicity in mice and a neurological condition called barrel rotation syndrome. In the present work, we report the molecular cloning of five new nucleotide sequences from a cDNA library of the venom glands of a single specimen of C. d. terrificus. These sequences have been analyzed in silico with respect to their cDNA organization and similarity with other snake venom serine proteases (SVSPs). We also describe a rapid and efficient method for screening vectors for mammalian cell expression, based on the fact that SVSPs are difficult-to-express toxins due to the presence of several disulfide bonds and glycosylation in their structures. Thus, one of the Gyroxin cDNAs was subcloned into pSectag2 HygroA and pED vectors and used to transfect COS-7 cells. Expression of the functional recombinant Gyroxin isoform was achieved with this cell line with esterase activity in the conditioned culture medium, as revealed by immunoblot of secreted protein and standard anti-crotalic serum from Butantan Institute.

High-level synthesis of recombinant murine endostatin in Chinese hamster ovary cells.

Endostatin, a carboxy-terminal fragment of collagen XVIII, has been shown to act as an anti-angiogenic agent that specifically inhibits proliferation of endothelial cells and growth of various primary tumors. Here, we describe the expression by Chinese hamster ovary (CHO) cells of murine endostatin and of a tagged-fusion protein, (his)6-met-endostatin. A dicistronic mRNA expression vector was utilized in which endostatin cDNA was inserted upstream of the amplifiable marker gene, dihydrofolate reductase (DHFR). After transfection of the expression vectors, stepwise increments in methotrexate levels in the culture medium were applied, promoting gene amplification and increasing expression levels of the proteins of interest. The expression level of secreted native endostatin was about 78 microg/mL while the one for secreted (his)6-met-endostatin was about 114 microg/mL, for the best expressing clones. Characterization of physico-chemical and immunological activities of the proteins was performed using SDS-PAGE and Western blotting. The biological activities of recombinant endostatins were tested with a cow pulmonary artery endothelial (C-PAE) cell proliferation assay. Both recombinant endostatin and (his)6-met-endostatin inhibited, in a dose-dependent fashion, growth of C-PAE cells stimulated by basic fibroblast growth factor (bFGF).

Current and prospective applications of metal ion-protein binding.

Since immobilized metal ion affinity chromatography (IMAC) was first introduced, several variants of this method and many other metal affinity-based techniques have been devised. IMAC quickly established itself as a highly reliable purification procedure, showing rapid expansion in the number of preparative and analytical applications while not remaining confined to protein separation. It was soon applied to protein refolding (matrix-assisted refolding), evaluation of protein folding status, protein surface topography studies and biosensor development. In this review, applications in protein processing are described of IMAC as well as other metal affinity-based technologies.

Ni(II)-based immobilized metal ion affinity chromatography of recombinant human prolactin from periplasmic Escherichia coli extracts.

A novel, two-step preparative technique is described for the purification of authentic recombinant human prolactin (rhPRL) secreted into the periplasm of transformed Escherichia coli cells. The first step is based on immobilized metal ion affinity chromatography of periplasmic extract, using Ni(II) as a relatively specific ligand for hPRL in this system. It gives superior resolution and yield than established ion-exchange chromatography. Size-exclusion chromatography is used for further purification to >99.5% purity. The methodology is reproducible, leading to 77% recovery. Identity and purity of the rhPRL were demonstrated using sodium dodecylsulphate-polyacrylamide electrophoresis, isoelectric focusing, mass spectrometry (matrix-assisted laser desorption ionization time-of-flight), radioimmunoassay, RP-HPLC and high-performance size-exclusion chromatography. In the Nb2 bioassay, the hormone showed a bioactivity of 40.9 IU/mg.

High-level synthesis of human prolactin in Chinese-Hamster ovary cells.

Two eukaryotic human prolactin (hPRL) expression vectors, based on a selectable dihydrofolate reductase (dhfr) marker, were used to transfect dhfr(-) Chinese- hamster ovary (CHO) cells. One vector, p658-hPRL, contains the hepatitis-B virus-X cDNA coding for a viral transactivator and sequences mediating dhfr mRNA degradation. The other, pEDdc-hPRL, carries the encephalomyocarditis virus leader sequence coupled to hPRL cDNA to provide high-level protein expression, possibly via a mechanism of internal translation initiation in dicistronic mRNA. Without methotrexate (MTX) amplification, p658-hPRL-transfected stable cell lines, secreting up to approximately 10 microg of hPRL/10(6) cells per day, could be rapidly obtained; production by pEDdc-hPRL-transfected cells was about 10-fold lower. However, a three-step MTX amplification of the latter led to clones secreting up to approximately 30 microg of hPRL/10(6) cells per day. A pilot production using a hollow-fibre bioreactor indicated that highly concentrated hormone levels in the medium could be obtained, with a production of up to 150 microg of hPRL/ml per day. SDS/PAGE analysis indicated that recombinant hPRL contained approximately 10% glycosylated PRL. Chromatographically purified non-glycosylated and glycosylated recombinant hPRL had bioactivities of 35 and 16 i.u./mg, respectively (Nb2 cell bioassay). This appears to be the first report describing production and purification of recombinant hPRL from CHO cells, secreted at levels higher than reported thus far in eukaryotic systems.

High-yield purification of biosynthetic human growth hormone secreted in Escherichia coli periplasmic space.

A six-step, high-yield purification procedure for the preparation of clinical grade recombinant human growth hormone (rhGH) secreted in bacterial periplasmic space is described. Particular emphasis is given to hormone recovery yields and maximum contaminant host cell elimination. The strategy adopted, in addition to using one precipitation and five chromatographic steps in a particularly efficient sequence, was also based on running E. coli proteins - immunoradiometric assay profiles right after each chromatographic elution. Thus, an overall rhGH recovery higher than 40%, with a final concentration of E. coli proteins below 10 ppm is described for the first time. The accuracy of hGH and total protein quantification, especially in the early steps of the process, and the maximum elimination of hGH-related forms were also studied in detail. For these purposes size-exclusion and reversed-phase HPLC were found to be extremely valuable analytical tools.

Synthesis and characterization of recombinant, authentic human prolactin secreted into the periplasmic space of Escherichia coli.

Recombinant, fully bioactive, authentic human prolactin (aut-hPRL) has been synthesized in transformed Escherichia coli HB2151 bacteria in a soluble, non-glycosylated form, which is secreted into the bacterial periplasm. Use was made of a bacterial expression vector, containing tac promoter-controlled sequences for the translation enhancer from bacteriophage T7 gene 10, and for a cellulase leader peptide from Cellulomonas fimi joined to sequences coding for aut-hPRL. This vector was derived from a previously described vector containing sequences of an hPRL variant, tag-hPRL (containing a 12-amino-acid peptide tag at the N-terminal end), using site-specific mutagenesis to delete the tag sequence. SDS/PAGE, partial N-terminal amino acid sequence analysis, Western blot analysis and Nb2 lymphoma cell in vitro bioassay indicated correct processing of the hormone. Periplasmic secretion of aut-hPRL, as measured by immunoassay, was relatively low (approx. 0.08 microgram/ml per A600 unit), in contrast to that of tag-hPRL which was approximately 8-fold higher, apparently a consequence of the tag sequence. This is the first report describing periplasmic secretion of biologically active, authentic hPRL.

Production and characterization of biologically active Ala-Ser-(His)6-Ile-Glu-Gly-Arg-human prolactin (tag-hPRL) secreted in the periplasmic space of Escherichia coli.

Human prolactin (hPRL) cDNA was obtained by screening of a pituitary cDNA library with a synthetic 21-mer oligonucleotide and with rat PRL cDNA. For its expression, use was made of a vector, p3SN8, containing tac-promoter-controlled sequences for a bacterial cellulase leader joined to sequences coding for Ala-Ser, a chromatographic affinity site consisting of six histidines and a Factor Xa cleavage site. The hPRL cDNA was inserted at the 3' end of the cleavage-site sequences. Expression in Escherichia coli led to secretion in the periplasmic space of a fully bioactive hPRL variant constituting authentic hPRL with a peptide tag, i.e. Ala-Ser-(His)6-Ile-Glu-Gly-Arg, at its N-terminal. This tag-hPRL could be rapidly and efficiently purified by metal-chelate affinity chromatography. The correct processing and quality of tag-hPRL was monitored by SDS/PAGE, Western-blot analysis, immunoassay and Nb2-lymphoma-cell bioassay. Treatment with Factor Xa for tag removal was only partially successful. Periplasmic secretion of tag-hPRL of the order of 0.7 micrograms/ml per A600 unit and one-step purification indicate feasibility for tag-hPRL production for in vitro diagnostic and research applications. This is the first report describing periplasmic secretion of a bioactive form of hPRL.

The use of recombinant human growth hormone for radioiodination and standard preparation in radioimmunoassay.

Recombinant human growth hormone (rec-hGH) obtained by cloning hGH precursor gene, bacterial expression and periplasmic secretion of the authentic, mature form of the hormone was used, after purification and characterization, for the preparation of radioimmunoassay (RIA) reagents. 125I-rec-hGH was prepared by the classical chloramine-T iodination technique, while an internal standard of the same rec-hGH was used and calibrated against pituitary hGH reference preparation (NIDDK-hGH-RP-1) with the use of a reference antiserum (NIDDK-anti-hGH-2). In both cases the behavior of the recombinant preparation was identical to that of the pituitary hormone. This confirms previous data on bacterial correct processing and folding of the protein, as far as its immunological behavior is concerned and indicates its suitability for the preparation of immunoassay reagents.

Morphological and biochemical variability of Microsporum canis strains.

Seventy-two strains of Microsporum canis, of different origins, were examined from a morphological point of view and tested in relation to their hydrolytic activity on tyrosine, xanthine, casein, gelatin, their ureasic activity and their capacity to assimilate different nitrogenous substances. The morphological aspects, that vary within the M. canis isolates, were constant in the strains isolated from rabbits. A strain with particular features was isolated many times from the dogs and cats coming from the same breeder. In one case of pseudomycetoma, different isolates suggested the co-existence in animals of two different strains, one present on fur, the other responsible for deep lesions.