Evaluation of a novel fusion system for soluble protein overexpression in Escherichia coli

Proteins production requires a successful correlation between expression, solubility and purification steps. As an expression system, Escherichia coli combines its low cost and ease of use with rapid expression, being widely used for heterologous protein production. This host cell has however several drawbacks, namely at the expression of insoluble proteins aggregated into inclusion bodies. Many efforts have been made to overcome such problems, including the optimization of expression conditions and the use of solubility fusion tags. The use of fusion tags for protein production remains challenging since none of the available fusion systems work universally with every partner protein. A novel fusion system had been recently discovered and submitted to a patenting process by Hitag Biotechnology, Lda. This fusion system consists of low molecular weight peptides/proteins from recombinant antigens, which have demonstrated to increase soluble protein expression levels in E. coli. This work aims at the evaluation of the effects of two novel fusion tags on soluble protein expression in E. coli. Specific primers were designed to amplify and sub‐clone gene sequences that encodes for frutalin, Cryptosporidium parvum 12kDa protein and Giardia lamblia cyst wall protein. These target proteins present therapeutic and diagnostic interests and had shown to be difficult‐to‐express in E. coli. Proteins were first fused to novel tags and than expressed in E. coli. Proteins purification was carried out by affinity chromatography, using nickel‐NTA columns. Pooled fractions were dialysed against phosphate buffer pH 7.4 and latter analysed by SDS‐PAGE. Protein expression levels were determined by Bradford assay. When fused to novel tags, all target proteins were successfully expressed in E. coli. Comparing to the respective non‐fused proteins, both novel tags used in this work promoted an increase from three to nine folds on soluble proteins expression levels. The SDS‐PAGE analysis confirmed the purity of Ni‐NTA pooled fractions, corroborating also these results. Tag1‐fusions achieved higher production yields than fusions with Tag2. In this work, three different target proteins were used to evaluate two novel fusion tags. The soluble overexpression effect offered by this novel fusion system may provide an important advance in recombinant protein expression processes in E. coli

A novel adjuvant-free H fusion system for the production of recombinant immunogens in Escherichia coli : Its application to a 12 kDa antigen from Cryptosporidium parvum

The production of recombinant antigens in Escherichia coli and specific polyclonal antibodies for diagnosis and therapy is still a challenge for world-wide researchers. Several different strategies have been explored to improve both antigen and antibody production, all of them depending on a successful expression and immunogenicity of the antigen. Gene fusion technology attempted to address these challenges: fusion partners have been applied to optimise recombinant antigen production in E. coli, and to increase protein immunogenicity. Taking a 12-kDa surface adhesion antigen from Cryptosporidium parvum (CP 12) by example, the novel H fusion partner was presented in this work as an attractive option for the development of recombinant immunogens and its adjuvant-free immunisation. The H tag (of only 1 kDa) efficiently triggered a CP 12-specific immune response, and it also improved the immunisation procedure without requiring coadministration of adjuvants. Moreover, polyclonal antibodies raised against the HCP 12 fusion antigen detected native antigen structures displayed on the surface of C. parvum oocysts. The H tag proved to be an advanced strategy and promising technology for the diagnosis and therapy of C. parvum infections in animals and humans, allowing a rapid and simple recombinant production of the CP 12 antigen.The financial support of Fundacao para a Ciencia e Tecnologia (FCT), Portugal, is acknowledged: Project PTDC/CVT/103081/2008 (co-financed by COMPETE) and grant SFRH/BD/46482/2008 (POPH-QREN) to Costa SJ. We would like to thank Lurdes Delgado and Sonia Soares for the Cryptosporidium oocysts isolation from fecal samples, and also to Hitag (R) Biotechnology, Ltd for kindly providing the H and Fh8 tag sequences used in this work

Zinc accumulation in Solanum Nigrum is enhanced by different arbuscular mycorrhizal fungi

Solanum nigrum was found to proliferate in sediments with high levels of metal pollution. The effect of Zn on plant growth and tissue metal accumulation was assessed. The response of the plant to the inoculation with four different isolates of arbuscular mycorrhizal fungi (AMF) (Glomus sp. BEG140, Glomus claroideum, Glomus mosseae and Glomus intraradices) was studied. While the isolates of AMF did not have a significant (P < 0.05) influence on mycorrhizal colonisation, increasing Zn concentration to high levels (500 and 1000 mg kg 1) induced significant (P < 0.05) decrease of the AMF colonisation. In general, the presence of AMF did not affect the growth and biomass of S. nigrum individuals. However, the level of metal in the matrix affected S. nigrum growth; plants grown at 100 mg kg 1 had significantly (P < 0.05) lower leaf, stem, root and total biomass than control ones and plants growing at 500 and 1000 mg kg 1 had the significantly (P < 0.05) lowest biomass. Plants inoculated with the AMF G. claroideum and G. intraradices presented significantly (P < 0.05) higher Zn accumulation in all plant tissues. In general, the stem tissues had the higher Zn content while the leaves registered the lowest values, which indicate a high translocation of the metal. AMF inoculation had no significant (P < 0.05) influence on the metal translocation within the plant. This study suggests that inoculation with the AMF G. claroideum or G. intraradices, can enhance the Zn accumulation in the tissues of S. nigrum, not affecting the plant translocation capacities

Zinc accumulation in plant species indigenous to a portuguese polluted site: relation with soil contamination

The levels of zinc accumulated by roots, stems, and leaves of two plant species, Rubus ulmifolius and Phragmites australis, indigenous to the banks of a stream in a Portuguese contaminated site were investigated in field conditions. R. ulmifolius, a plant for which studies on phytoremediation potential are scarce, dominated on the right side of the stream, while P. australis proliferated on the other bank. Heterogeneous Zn concentrations were found along the banks of the stream. Zn accumulation in both species occurred mainly in the roots, with poor translocation to the aboveground sections. R. ulmifolius presented Zn levels in the roots ranging from 142 to 563 mg kg21, in the stems from35 to 110 mg kg21, and in the leaves from 45 to 91 mg kg21, vs. average soil total Zn concentrations varying from 526 to 957 mg kg21. P. australis showed Zn concentrations in the roots from 39 to 130 mg kg21, in the stems from 31 to 63 mg kg21, and in the leaves from 37 to 83 mg kg21, for the lower average soil total Zn levels of 138 to 452 mg kg21 found on the banks where they proliferated. Positive correlations were found between the soil total, available and extractable Zn fractions, and metal accumulation in the roots and leaves of R. ulmifolius and in the roots and stems of P. australis. The use of R. ulmifolius and P. australis for phytoextraction purposes does not appear as an effective method of metal removing, but these native metal tolerant plant species may be used to reduce the effects of soil contamination, avoiding further Zn transfer to other environmental compartment

The Fh8 tag : a fusion partner for simple and cost-effective protein purification in Escherichia coli

Downstream processing is still a major bottleneck in recombinant protein production representing most of its costs. Hence, there is a continuing demand of novel and cost-effective purification processes aiming at the recovery of pure and active target protein. In this work, a novel purification methodology is presented, using the Fh8 solubility enhancer tag as fusion handle. The binding properties of Fh8 tag to a hydrophobic matrix were first studied via hydrophobic interaction chromatography (HIC). The Fh8 tag was then evaluated as a purification handle by its fusion to green fluorescent protein and superoxide dismutase. The purification efficiency of the Fh8-HIC strategy was compared to the immobilized metal ion affinity chromatography (IMAC) using the His6 tag. Results showed that the Fh8-HIC binding mechanism is calcium-dependent in a low salt medium, making the purification process highly selective. Both target proteins were biologically active, even when fused to Fh8, and were successfully purified by HIC, achieving efficiencies identical to those of IMAC. Thus, the Fh8 acts as an effective affinity tag that, together with its previously reported solubility enhancer capability, allows for the design of inexpensive and successful recombinant protein production processes in Escherichia coli.This work was conducted with the financial support of the Fundacao para a Ciencia e Tecnologia (FCT), Portugal, by the fellowship SFRH/BD/46482/2008 (POPH-QREN) to Sofia J. Costa. The FCT project PTDC/CVT/103081/2008 (co-funded by COMPETE) and QREN co-promotion project number 3515 are also acknowledged. The authors thank Dr. Huseyin Besir, Protein Expression and Purification Facility Core, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany, for supplying the pETM11 expression vector, and also Dr. Vitor Costa for kindly providing the SOD target gene used in this work