A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli.

Monoclonal antibodies (mABs) are of great biopharmaceutical importance for the diagnosis and treatment of diseases. However, their production in mammalian expression hosts usually requires extensive production times and is expensive. Escherichia coli has become a new platform for production of functional small antibody fragment variants. In this study, we have used a rhamnose-inducible expression system that allows precise control of protein expression levels. The system was first evaluated for the cytoplasmic production of super folder green fluorescence protein (sfGFP) in various production platforms and then for the periplasmic production of the anti-HIV single-chain variable antibody fragment (scFv) of PGT135. Anti-HIV broadly neutralizing antibodies, like PGT135, have potential for clinical use to prevent HIV transmission, to promote immune responses and to eradicate infected cells. Different concentrations of L-rhamnose resulted in the controlled production of both sfGFP and scFv PGT135 antibody. In addition, by optimizing the culture conditions, the amount of scFv PGT135 antibody that was expressed soluble or as inclusions bodies could be modulated. The proteins were produced in batch bioreactors, with yields of 4.9 g/L for sfGFP and 0.8 g/L for scFv. The functionality of the purified antibodies was demonstrated by their ability to neutralize a panel of different HIV variants in vitro. We expect that this expression system will prove very useful for the development of a more cost-effective production process for proteins and antibody fragments in microbial cells.

A Periplasmic Complex of the Nitrite Reductase NirS, the Chaperone DnaK, and the Flagellum Protein FliC Is Essential for Flagellum Assembly and Motility in Pseudomonas aeruginosa.

UNLABELLED: Pseudomonas aeruginosa is a ubiquitously occurring environmental bacterium and opportunistic pathogen responsible for various acute and chronic infections. Obviously, anaerobic energy generation via denitrification contributes to its ecological success. To investigate the structural basis for the interconnection of the denitrification machinery to other essential cellular processes, we have sought to identify the protein interaction partners of the denitrification enzyme nitrite reductase NirS in the periplasm. We employed NirS as an affinity-purifiable bait to identify interacting proteins in vivo. Results obtained revealed that both the flagellar structural protein FliC and the protein chaperone DnaK form a complex with NirS in the periplasm. The interacting domains of NirS and FliC were tentatively identified. The NirS-interacting stretch of amino acids lies within its cytochrome c domain. Motility assays and ultrastructure analyses revealed that a nirS mutant was defective in the formation of flagella and correspondingly in swimming motility. In contrast, the fliC mutant revealed an intact denitrification pathway. However, deletion of the nirF gene, coding for a heme d1 biosynthetic enzyme, which leads to catalytically inactive NirS, did not abolish swimming ability. This pointed to a structural function for the NirS protein. FliC and NirS were found colocalized with DnaK at the cell surface of P. aeruginosa. A function of the detected periplasmic NirS-DnaK-FliC complex in flagellum formation and motility was concluded and discussed. IMPORTANCE: Physiological functions in Gram-negative bacteria are connected with the cellular compartment of the periplasm and its membranes. Central enzymatic steps of anaerobic energy generation and the motility mediated by flagellar activity use these cellular structures in addition to multiple other processes. Almost nothing is known about the protein network functionally connecting these processes in the periplasm. Here, we demonstrate the existence of a ternary complex consisting of the denitrifying enzyme NirS, the chaperone DnaK, and the flagellar protein FliC in the periplasm of the pathogenic bacterium P. aeruginosa. The dependence of flagellum formation and motility on the presence of an intact NirS was shown, structurally connecting both cellular processes, which are important for biofilm formation and pathogenicity of the bacterium.

The 3,4-dihydroxyphenylacetic acid catabolon, a catabolic unit for degradation of biogenic amines tyramine and dopamine in Pseudomonas putida U.

Degradation of tyramine and dopamine by Pseudomonas putida U involves the participation of twenty one proteins organized in two coupled catabolic pathways, Tyn (tynABFEC tynG tynR tynD, 12 338 bp) and Hpa (hpaR hpaBC hpaHI hpaX hpaG1G2EDF hpaA hpaY, 12 722 bp). The Tyn pathway catalyses the conversion of tyramine and dopamine into 4-hydroxyphenylacetic acid (4HPA) and 3,4-dihydroxyphenylacetic acid (3,4HPA) respectively. Together, the Tyn and Hpa pathways constitute a complex catabolic unit (the 3,4HPA catabolon) in which 3,4HPA is the central intermediate. The genes encoding Tyn proteins are organized in four consecutive transcriptional units (tynABFEC, tynG, tynR and tynD), whereas those encoding Hpa proteins constitute consecutive operons (hpaBC, hpaG1G2EDF, hpaX, hpaHI) and three independent units (hpaA, hpaR and hpaY). Genetic engineering approaches were used to clone tyn and hpa genes and then express them, either individually or in tandem, in plasmids and/or bacterial chromosomes, resulting in recombinant bacterial strains able to eliminate tyramine and dopamine from different media. These results enlarge our biochemical and genetic knowledge of the microbial catabolic routes involved in the degradation of aromatic bioamines. Furthermore, they provide potent biotechnological tools to be used in food processing and fermentation as well as new strategies that could be used for pharmacological and gene therapeutic applications in the near future.

Ex Vivo Expansion of Hematopoietic Stem Cells for Therapeutic Purposes: Lessons from Development and the Niche.

Expansion of hematopoietic stem cells (HSCs) for therapeutic purposes has been a "holy grail" in the field for many years. Ex vivo expansion of HSCs can help to overcome material shortage for transplantation purposes and genetic modification protocols. In this review, we summarize improved understanding in blood development, the effect of niche and conservative signaling pathways on HSCs in mice and humans, and also advances in ex vivo culturing protocols of human HSCs with cytokines or small molecule compounds. Different expansion protocols have been tested in clinical trials. However, an optimal condition for ex vivo expansion of human HSCs still has not been found yet. Translating and implementing new findings from basic research (for instance by using genetic modification of human HSCs) into clinical protocols is crucial to improve ex vivo expansion and eventually boost stem cell gene therapy.

Genetic analyses and molecular characterization of the pathways involved in the conversion of 2-phenylethylamine and 2-phenylethanol into phenylacetic acid in Pseudomonas putida U.

In Pseudomonas putida U two different pathways (Pea, Ped) are required for the conversion of 2-phenylethylamine and 2-phenylethanol into phenylacetic acid. The 2-phenylethylamine pathway (PeaABCDEFGHR) catalyses the transport of this amine, its deamination to phenylacetaldehyde by a quinohaemoprotein amine dehydrogenase and the oxidation of this compound through a reaction catalysed by a phenylacetaldehyde dehydrogenase. Another catabolic route (PedS(1)R(1)ABCS(2)R(2)DEFGHI) is needed for the uptake of 2-phenylethanol and for its oxidation to phenylacetic acid via phenylacetaldehyde. This implies the participation of two different two-component signal-transducing systems, two quinoprotein alcohol dehydrogenases, a cytochrome c, a periplasmic binding protein, an aldehyde dehydrogenase, a pentapeptide repeat protein and an ABC efflux system. Additionally, two accessory sets of elements (PqqABCDEF and CcmABCDEFGHI) are necessary for the operation of the main pathways (Pea and Ped). PqqABCDEF is required for the biosynthesis of pyrroloquinoline quinone (PQQ), a prosthetic group of certain alcohol dehydrogenases that transfers electrons to an independent cytochrome c; whereas CcmABCDEFGHI is required for cytochrome c maturation. Our data show that the degradation of phenylethylamine and phenylethanol in P. putida U is quite different from that reported in Escherichia coli, and they demonstrate that PeaABCDEFGHR and PedS(1)R(1)ABCS(2)R(2)DEFGHI are two upper routes belonging to the phenylacetyl-CoA catabolon.

A genetically engineered strain of Pseudomonas putida as a useful tool for identifying new therapeutic herbicides.

A genetically engineered strain of Pseudomonas putida U designed for the identification of new therapeutic herbicides has been obtained. In this bacterium, deletion of the homogentisate gene cluster (hmgRABC) confers upon this mutant huge biotechnological possibilities since it can be used: (i) as a target for testing new specific herbicides (p-hydroxy-phenylpyruvate dioxygenase inhibitors); (ii) to identify new therapeutic drugs-effective in the treatment of alkaptonuria and other related tyrosinemia - and (iii) as a source of homogentisic acid in a plant-bacterium association.

Tight coupling of polymerization and depolymerization of polyhydroxyalkanoates ensures efficient management of carbon resources in Pseudomonas putida.

Environmental microbes oscillate between feast and famine and need to carefully manage utilization, storage and conversion of reserve products to exploitable sources of carbon and energy. Polyhydroxyalkanoates (PHAs) are storage polymers that serve bacteria as sources of food materials under physiological conditions of carbon demand. In order to obtain insights into the role of PHA depolymerase (PhaZ) and its relationship to a PHA polymerase (PhaC2) in the carbon management activity of Pseudomonas putida strain U, we created a polymerase hyperexpression strain and a depolymerase knockout mutant of this strain, and examined their synthesis of PHA and expression of their PHA genes. This study revealed that hyperexpression of PhaC2 led to the accumulation of higher amounts of PHA (44%wt) than in the wild-type strain (24%wt) after 24 h of cultivation, which then returned to wild-type levels by 48 h, as a result of elevated depolymerization. The phaZ mutant, however, accumulated higher levels of PHA than the parental strain (62%wt), which were maintained for at least 96 h. Transcriptional analysis of the pha cluster by RT-PCR revealed that PHA operon proteins, including depolymerase, are expressed from the beginning of the growth phase. Hyperexpression of the PhaC2 polymerase was accompanied by an increase in the expression of the PhaZ depolymerase and a decrease in expression of another PHA polymerase, PhaC1. This suggests tight regulatory coupling of PHA polymerase and depolymerase activities that act in synergy, and in concert with other PHA proteins, to provide dynamic PHA granule synthesis and remodelling that rapidly and sensitively respond to changes in availability of carbon and the physiological-metabolic needs of the cell, to ensure optimal carbon resource management.

Exploiting metagenomic diversity for novel polyhydroxyalkanoate synthases: production of a terpolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate) with a recombinant Pseudomonas putida strain.

A metagenomic library of 2.1×10(6) clones was constructed using oil-contaminated soil from Gujarat (India). One of the fosmid clones, 40N22, encodes a polyhydroxyalkanoate synthase showing 76% identity with an Alcaligenes sp. synthase. The corresponding gene was expressed in Pseudomonas putida KT2440 ΔphaC1 which is impaired in PHA production. The gene conferred the recombinant strain PpKT-40N22 with the ability to produce copolymers with up to 21% in medium-chain-length content. Thus, 37% and 45% of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate), respectively were obtained when using sodium heptanoate and oleic acid as carbon sources. These 3-hydroxybutyrate-(3HB)-based polymers are of interest since they incorporate the properties of medium chain length polymers and thus increase the range of applications of PHAs.

Characterisation of the putative effector interaction site of the regulatory HbpR protein from Pseudomonas azelaica by site-directed mutagenesis.

Bacterial transcription activators of the XylR/DmpR subfamily exert their expression control via σ(54)-dependent RNA polymerase upon stimulation by a chemical effector, typically an aromatic compound. Where the chemical effector interacts with the transcription regulator protein to achieve activation is still largely unknown. Here we focus on the HbpR protein from Pseudomonas azelaica, which is a member of the XylR/DmpR subfamily and responds to biaromatic effectors such as 2-hydroxybiphenyl. We use protein structure modeling to predict folding of the effector recognition domain of HbpR and molecular docking to identify the region where 2-hydroxybiphenyl may interact with HbpR. A large number of site-directed HbpR mutants of residues in- and outside the predicted interaction area was created and their potential to induce reporter gene expression in Escherichia coli from the cognate P(C) promoter upon activation with 2-hydroxybiphenyl was studied. Mutant proteins were purified to study their conformation. Critical residues for effector stimulation indeed grouped near the predicted area, some of which are conserved among XylR/DmpR subfamily members in spite of displaying different effector specificities. This suggests that they are important for the process of effector activation, but not necessarily for effector specificity recognition.

Poly-3-hydroxyalkanoate synthases from Pseudomonas putida U: substrate specificity and ultrastructural studies.

The substrate specificity of the two polymerases (PhaC1 and PhaC2) involved in the biosynthesis of medium-chain-length poly-hydroxyalkanoates (mcl PHAs) in Pseudomonas putida U has been studied in vivo. For these kind of experiments, two recombinant strains derived from a genetically engineered mutant in which the whole pha locus had been deleted (P. putida U Δpha) were employed. These bacteria, which expresses only phaC1 (P. putida U Δpha pMC-phaC1) or only phaC2 (P. putida U Δpha pMC-phaC2), accumulated different PHAs in function of the precursor supplemented to the culture broth. Thus, the P. putida U Δpha pMC-phaC1 strain was able to synthesize several aliphatic and aromatic PHAs when hexanoic, heptanoic, octanoic decanoic, 5-phenylvaleric, 6-phenylhexanoic, 7-phenylheptanoic, 8-phenyloctanoic or 9-phenylnonanoic acid were used as precursors; the highest accumulation of polymers was observed when the precursor used were decanoic acid (aliphatic PHAs) or 6-phenylhexanoic acid (aromatic PHAs). However, although it synthesizes similar aliphatic PHAs (the highest accumulation was observed when hexanoic acid was the precursor) the other recombinant strain (P. putida U Δpha pMC-phaC2) only accumulated aromatic PHAs when the monomer to be polymerized was 3-hydroxy-5-phenylvaleryl-CoA. The possible influence of the putative three-dimensional structures on the different catalytic behaviour of PhaC1 and PhaC2 is discussed.

Strategy for cloning large gene assemblages as illustrated using the phenylacetate and polyhydroxyalkanoate gene clusters.

We report an easy procedure for isolating chromosome-clustered genes. By following this methodology, the entire set of genes belonging to the phenylacetic acid (PhAc; 18-kb) pathway as well as those required for the synthesis and mobilization of different polyhydroxyalkanoates (PHAs; 6.4 kb) in Pseudomonas putida U were recovered directly from the bacterial chromosome and cloned into a plasmid for the first time. The transformation of different bacteria with these genetic constructions conferred on them the ability to either degrade PhAc or synthesize bioplastics (PHAs).