isopropyl-beta-D-thiogalactoside Research Articles

Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases.

Directed evolution and targeted genome editing have been deployed to create genetic variants with usefully altered phenotypes. However, these methods are limited to high-throughput screening methods or serial manipulation of single genes. In this study, we implemented multicopy chromosomal integration using CRISPR-associated transposases (MUCICAT) to simultaneously target up to 11 sites on the Escherichia coli chromosome for multiplex gene interruption and/or insertion, generating combinatorial genomic diversity. The MUCICAT system was improved by replacing the isopropyl-beta-D-thiogalactoside (IPTG)-dependent promoter to decouple gene editing and product synthesis and truncating the right end to reduce the leakage expression of cargo. We applied MUCICAT to engineer and optimize the N-acetylglucosamine (GlcNAc) biosynthesis pathway in E. coli to overproduce the industrially important GlcNAc in only 8 days. Two rounds of transformation, the first round for disruption of two degradation pathways related gene clusters and the second round for multiplex integration of the GlcNAc gene cassette, would generate a library with 1-11 copies of the GlcNAc cassette. We isolated a best variant with five copies of GlcNAc cassettes, producing 11.59 g/L GlcNAc, which was more than sixfold than that of the strain containing the pET-GNAc plasmid. Our multiplex approach MUCICAT has potential to become a powerful tool of cell programing and can be widely applied in many fields such as synthetic biology.

Expression and purification of Rv2654 recombinant protein from Mycobacterium tuberculosis and its characteristics of immune response.

With the decreased protective effect of Bacille Calmette-Guérin (BCG) vaccine, widespread drug-resistant strains of tuberculosis as well as the lack of effective vaccines, global morbidity and mortality of tuberculosis remains high. The purpose of this study is to evaluate the potential of Mycobacterium tuberculosis antigen Rv2654 as a candidate vaccine against tuberculosis, and to verify the characteristics of cellular and humoral immune responses in mice induced by this protein. Isopropyl-beta-D-thiogalactoside (IPTG) was added to induce the expression of Rv2654 recombinant protein in the logarithmic growth stage of bacteria. The recombinant protein was purified by affinity chromatography and identified by Western blotting. The immunoreactivity of Rv2654 recombinant protein with human sera was detected by ELISA. After immunization with Rv2654 recombinant protein, the levels of Th1/Th2 cytokines in peripheral blood of mice was measured using cytokine magnetic bead arrays, and the T and B lymphocyte subsets in spleen of mice was analyzed by flow cytometry. The recombinant protein Rv2654 was successfully induced and expressed. The ELISA data showed that the recombinant protein Rv2654 was responsive to the serum of BCG-inoculated patients or active pulmonary tuberculosis patients. In immunized mice with recombinant protein Rv2654, the expression of IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10 and other cytokines in peripheral blood was elevated (all P<0.05). Flow cytometry analysis showed that the recombinant protein significantly stimulated the differentiation of CD4+T cells and CD8+T cells into effector T cells, and this effect was more obvious when combined with BCG. The recombinant protein Rv2654 also stimulated the activation and proliferation of B cells and their differentiation into memory cells. However, less plasma cells were produced. Rv2654 protein could induce the cell immune responses and it has good binding ability with serum of BCG-inoculated patients and active pulmonary tuberculosis patients, suggesting that it may serve as a good candidate antigen for tuberculosis immunological prophylaxis and diagnostic vaccines.

Optimization of the expression of phaC2 encoding poly (3-hydroxyalkanoate) synthase from Pseudomonas aeruginosa PTCC1310 in Fad B deleted Escherichia coli.

Background:Poly3-hydroxyalkanoates (PHAs) are potential candidates for the industrial production of biodegradable plastics. Therefore, in the present study, expression and activity of one of the enzymes involved in the PHA synthesis, phaC2 (isolated from Pseudomonas aeruginosa PTCC1310), were investigated in Fad B deleted Escherichia coli.Materials and Methods:The inserts obtained from recombinant pTZ57R plasmids were ligated into the pGEX-5x-1 expression vector and then transformed into Fad B deleted E. coli cells using the heat shock method. This protein was then expressed using isopropyl beta-d-thiogalactoside (IPTG) as an inducer. By changing expression conditions such as IPTG and glucose concentration, time and temperature of incubation with IPTG, the expression conditions were optimized.Results:The optimum condition for the expression of this enzyme was: 1.5 mM IPTG, 1 mM glucose, incubated at 37°C for 2 hours.Conclusion:We obtained functional expression of the phaC2 gene and investigated various conditions that could influence the expression of protein to optimize production of PHA synthase enzymes. This would allow us to study PHA production in large quantities.

Constructing a synthetic metabolic pathway in Escherichia coli to produce the enantiomerically pure (R, R)-2,3-butanediol.

Enantiomerically pure (R, R)-2,3-butanediol has unique applications due to its special chiral group and spatial configuration. Currently, its chemical production route has many limitations. In addition, no native microorganisms can accumulate (R, R)-2,3-butanediol with an enantio-purity over 99%. Herein, we constructed a synthetic metabolic pathway for enantiomerically pure (R, R)-2,3-butanediol biosynthesis in Escherichia coli. The fermentation results suggested that introduction of the synthetic metabolic pathway redistributed the carbon fluxes to the neutral (R, R)-2,3-butanediol, and thus protected the strain against the acetic acid inhibition. Additionally, it showed that the traditionally used isopropyl beta-D-thiogalactoside (IPTG) induction displayed negative effect on (R, R)-2,3-butanediol biosynthesis in the recombinant E. coli, which was probably due to the protein burden. With no IPTG addition, the (R, R)-2,3-butanediol concentration reached 115 g/L by fed-batch culturing of the recombinant E. coli, with an enantio-purity over 99%, which is suitable for the pilot-scale production.

Expression and purification of an active cecropin-like recombinant protein against multidrug resistance Escherichia coli

Lucilin is a 36 residue cecropin antimicrobial peptide identified as a partial genetic sequence in Lucilia sericata maggots. The antimicrobial spectrum and toxicity profile of Lucilin is unknown. We first report the expression of Lucilin as an active recombinant fusion protein with a cysteine protease domain (CPD) tag. The fusion protein, GWLK-Lucilin-CPD-His8, showed maximum overexpression in Escherichia coli BL21 cells after 12h induction with 0.5mM IPTG (isopropyl beta-d-thiogalactoside) and growth conditions were 37°C and 150rpm shaking. The fusion protein was expressed as a soluble form and was purified by Ni-IMAC. The purified protein was active against E. coli ATCC 35218 with a MIC of 0.68μM, and a clinical isolate of E. coli with extended spectrum beta-lactamase (ESBL) with a MIC of 0.8μM. The recombinant GWLK-Lucilin-CPD-His8 was not toxic against human erythrocytes or Vero cells with a therapeutic index >63. The results suggest that GWLK-Lucilin-CPD-His8 represents a potential candidate for therapy against multidrug resistant Gram-negative bacteria.

Construction and use of a prokaryotic expression system for Helicobacter pylori AhpC

BackgroundHelicobacter pylori is an important pathogen responsible for human gastric problems like inflammation, ulcers and cancer. It is widely prevalent in developing countries with low socioeconomic status. Since the infection remains asymptomatic in most individuals, efforts for efficient diagnostic markers to identify high risk patients are warranted. In this study, we constructed an expression vector that overexpresses the H. pylori AhpC protein as a glutathione S-transferase fusion protein. We furthermore examined whether this recombinant fusion protein retained immunogenicity and thus would be useful as a diagnostic marker.FindingsThe full-length tsaA gene from H. pylori strain G27, which encodes AhpC, was cloned in plasmid vector pGEX-6P-2 to create the recombinant plasmid vector pGEX-tsaA. The nucleotide sequence of the clone showed 100% homology with corresponding published sequence of original gene. Over-expression of the target protein GST-AhpC was achieved in E. coli BL21 (DE3) cells by induction with isopropyl-beta-D-thiogalactoside (IPTG). GST-AhpC was extracted and identified using SDS-PAGE as a 52 kDa protein. Western blotting results using commercial antibodies against whole cell H. pylori showed that the fusion protein retained immunogenecity.ConclusionA recombinant prokaryotic expression system was successfully established with high expression efficiency for target fusion gene pGEX-tsaA. The expressed GST-AhpC protein showed immunoreactivity against commercial anti-H. pylori antibodies. This recombinant fusion protein can be developed as a diagnostic marker for screening patients with chronic H. pylori infections.

A two‐step fluorescence‐activated cell sorting approach to isolate genetically modified mammalian cells with isopropyl‐beta‐D‐thiogalactoside (IPTG)‐inducible gene expression

inducible gene expression system is a versatile tool tostudy protein function. It provides an alternative to transientsystems wherein constitutive over-expression of ectopic pro-teins can introduce deleterious effects such as cell cycle arrest(1) or apoptosis (2). The ability to switch off/on the expres-sion of a specific protein is particularly useful for comparativestudies using the same cell system. To date, a range of induci-ble systems have been described such as those responsive tometal (3), glucocorticoid (4), or isopropyl-beta-D-thiogalac-toside (IPTG) (5). Since metal ions and glucocorticoids areknown to affect the expression of many endogenous proteins,IPTG remains a more popular choice of inducer. Nevertheless,these systems share a common drawback in that generation ofinducible cell lines can be labor-intensive, time-consuming(up to several months), and inefficient (1–2 cell lines perstudy). Here we use the LacSwitch II Inducible MammalianExpression System (Agilent Technologies, Santa Clara, CA) asa typical model to describe an improved approach to generateinducible cell lines.The LacSwitch system is designed for inducible geneexpression in mammalian cells. This system employs two vec-tors, the pCMVLacI vector which confers hygromycin resist-ance and encodes Lac repressor, and the lac-operator-contain-ing pOPRSVI/MCS or pOPI3CAT vector which confers G418resistance and encodes the gene of interest. When both vectorsare transfected into a cultured cell line, the Lac protein isexpressed and binds to the lac-operators to repress expressionof the gene of interest. Such repression is constitutive until theinducer IPTG is added to the cell culture to inactivate the Lacprotein. There are different approaches to generate induciblecell lines using this system. While the transfection conditionsvary among different studies, the screening procedures areapparently consistent in that individual transfected clones aremanually isolated and expanded for examination of geneexpression by Western blot analysis. Since transfection effi-ciency and expression levels of ectopic proteins can be highlyvariable, it is often necessary to screen dozens to hundreds ofindividual clones to select the specific ones with desired induc-ible phenotypic characteristics. Although multiple methodssuch as cloning rings (6), trypsin-soaked filter circles (7), andserial dilution (8) are available to facilitate the isolation ofindividual clones, the screening process remains extremelylaborious and poses additional risks for tissue culture contam-ination. To address these concerns, we propose a two-stepapproach using fluorescent protein as a selection marker andfluorescence-activated cell sorting (FACS) to automate thescreening procedures.This FACS-based approach starts with co-transfection ofboth the pCMVLacI vector encoding Lac repressor and thepOPRSVI/MCS or pOPI3CAT vector encoding fluorescentprotein. Since transfection efficiency of individual vectors var-ies among cells, four types of clone variants are anticipatedwith integrated gene expressing (i) Lac only; (ii) fluorescentprotein only; (iii) both Lac and fluorescent protein; or (iv) noectopic protein, due to undesired genomic recombination.The IPTG-dependent expression profiles of individual clonesare summarized (Fig. 1A). Given the unique property of thedesired inducible type III clone, specific isolation is possibleby applying different combinations of induction and cell sort-ing criteria (Fig. 1B). The strategy is to first keep the mixed

Up-regulation of NKG2F receptor, a functionally unknown killer receptor, of human natural killer cells by interleukin-2 and interleukin-15

The NKG2 family receptors are C-type lectin, type II transmembrane molecules, and play important roles in regulation of natural killer (NK) cell functions against tumor and virus. NKG2F is a new member of NKG2 family, and may possibly associate with DAP 12 to activate NK cells. Since lacking available antibody against human NKG2F, the features of NKG2F expression on NK cells remains unclear. In this study, human NKG2F recombinant expression in E. coli was carried out by using pET-28a with a hexahistidine (6x His) tag and a thrombin digestion sequence to the N-terminus of the recombinant protein NKG2F. IPTG (isopropyl-beta-d-thio-galactoside) induction resulted in high expression of recombinant NKG2F protein, which was then purified and identified by anti-His western blotting and LC-MS/MS. Polyclonal antibody was produced by immunization of BALB/c mice with recombinant NKG2F, and then used to detect NKG2F in western blotting and flow cytometry. Our results demonstrated that NKG2F was expressed only by PBMCs but not by human NK cell lines such as NKL and YT at mRNA level. It was observed that NKG2F was expressed on surface of human blood NK cells, and may be up-regulated at mRNA level and protein level after IL-2 or IL-15 stimulation.

Optimization of an induction strategy for improving interferon‐α2b production in the periplasm of Escherichia coli using response surface methodology

Induction strategies for the periplasmic production of recombinant human IFN-alpha2b (interferon-alpha2b) by recombinant Escherichia coli Rosetta-gami 2(DE3) were optimized in shake-flask cultures using response surface methodology based on the central composite design. The factors included in the present study were induction point, which related to the attenuance of the cell culture, IPTG (isopropyl beta-D-thiogalactoside) concentration and induction temperature. Second-order polynomial models were used to correlate the abovementioned factors to soluble periplasmic IFN-alpha2b formation and percentage of soluble IFN-alpha2b translocated to the periplasmic space of E. coli. The models were found to be significant and subsequently validated. The proposed induction strategies consisted of induction at an attenuance of 4 (measured as D600), IPTG concentration of 0.05 mM and temperature of 25 degrees C. The optimized induction strategy reduced inclusion-body formation as evidenced by electron microscopy and yielded 323.8 ng/ml of IFN-alpha2b in the periplasmic space with translocation of 74% of the total soluble product. In comparison with the non-optimized condition, soluble periplasmic production and the percentage of soluble IFN-alpha2b translocated to the periplasmic space obtained in optimized induction strategies were increased by approx. 20-fold and 1.4-fold respectively.

A powerful hybrid puc operon promoter tightly regulated by both IPTG and low oxygen level

Rhodobacter sphaeroides has been intensively studied and provides an excellent model for studying both photosynthesis and membrane development. The photosynthetic apparatus (LH2 and LH1-RC complexes) can be synthesized in large scale and integrated into the intracytoplasmic membrane system under specific conditions, which thus provides us insight to utilize the puc or(and) puf operon to heterologously express recombinant proteins in the intracytoplasmic membrane using Rb. sphaeroides as a novel expression system. However, basal level of expression of puc and puf promoter is uncontrolled. We report the construction of LH2 polypeptide expression vector that contains a reengineered lacI(q)-puc promoter-lac operator hybrid promoter, which allows the puc operon to be regulated by both IPTG and low oxygen level. Synthesis of LH2 complexes was completely repressed in the absence of isopropyl beta-D-thiogalactoside (IPTG), and the degree of induction was controlled by varying the concentration of IPTG. The optimal concentration of IPTG was determined. SDS-PAGE and Western blot were employed for further analysis. Our results suggest that the reengineered hybrid promoter is efficient to tightly regulate the expression of the puc operon, and our strategy can open up a new approach in the study of the membrane protein expression system.

Cloning and expression of the membrane protein gene of TGEV HB06 strain.

The membrane protein gene(M) of transmissible gastroenteritis virus (TGEV) strain HB06, isolated from the feces of piglets infected with TGEVon a pig farm in Hebei province, was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). The amplified PCR products of TGEV HB06 were cloned, sequenced, and compared with other TGEV strains genes selected from the GenBank. Then, the recombinant fragment in pMD18-T was subcloned into corresponding sites of prokaryotic expression vector pGEX-6P-1 after digestion with EcoRI and XhoI to construct a recombinant fusion expression vector pGEX-6P-M. Then, the verified recombinant plasmid was transformed into Escherichia coli Rossetta (DE3), and the expression of M fusion protein was induced by using isopropylthio-beta-D-galactoside (IPTG) as inducer. The results showed that the gene fragment of M at a length of 789 bp was amplified and cloned into the vector pMD18-T successfully, and sequence comparison with that reported in GenBank revealed that the M gene complete sequence shares more than 94% homology in nucleotide. The result of SDS-PAGE showed that the recombinant membrane protein had a molecular mass of approximately 56 kDa, which was the same as the expected results. It was proven by Western blotting that the recombinant membrane protein had strong positive reactions with TGEV-specific antibody. Therefore, the expressed fusion protein has a good antigenicity. This work established a good foundation for further studies on the production of anti-TGEV vaccines.

Prokaryotic expression, purification and characterization of a novel pro-apoptosis protein hPNAS-4

Recently, hPNAS-4 [human PNAS-4; also known as C1orf121 (Genbank accession number NP-057160)] was reported to be a novel pro-apoptotic protein in mammalian cells. However, at present there is little information about hPNAS-4 and its molecular mechanisms. In our present studies, the hPNAS-4 gene was first cloned into the pGEX-6p-1 vector with a GST (glutathione transferase) tag to express in soluble form in Escherichia coli induced with 0.5 mM IPTG (isopropyl beta-D-thiogalactoside) at 25 degrees C, and the recombinant hPNAS-4 was purified to near homogeneity with 96% purity by affinity chromatography and anion-exchange chromatography. The purified hPNAS-4 protein was further identified by liquid chromatography-ESI (electrospray ionization)-MS analysis and used to raise polyclonal antibodies that could specifically recognize the hPNAS-4 protein. In addition, hPNAS-4 is localized to the cytoplasm and the perinuclear compartment. Furthermore, the overexpression of hPNAS-4 in human A549 lung cancer cells resulted in decrease of cell viability via apoptosis. This study represented an important step to investigate the characterization for the new pro-apoptosis protein hPNAS-4, which should aid the discovery of new drug targets for the development of effective therapeutic approaches to cancer in the future.

Cloning, Prokaryotic Expression, and Biological Analysis of Recombinant Chicken IFN-γ

The full-length chicken interferon-gamma (CHIFN-gamma) gene was amplified by reverse transcription-PCR using total RNA extracted from the spleen cells of white Leghorn chicken, a local Chinese breeding species. A truncated CHIFN-gamma gene without the N-terminal signal peptide sequence was cloned into prokaryotic expression vector pET30a, resulting in a recombinant plasmid pET-30a-CHIFN-gamma. After the recombinant plasmid was transformed into host cells BL21(DE3)pLysS, the expression of CHIFN-gamma was induced by isopropyl beta-D-thiogalactoside (IPTG). Rabbit antiserum was raised using the soluble CHIFN-gamma as immunogen. Immunoreactivities of the CHIFN-gamma and its antiserum were investigated using immunoblotting and ELISA. Moreover, the antiviral effect of the CHIFN-gamma was analyzed. Our data indicate that the CHIFN-gamma is biologically active.

Effects of feeding and induction strategy on the production ofBmR1 antigen in recombinantE. coli

To investigate the effects of feeding and induction strategies on the production of BmR1 recombinant antigen. Fed-batch fermentation was studied with respect to the specific growth rate and mode of induction to assess the growth potential of the bacteria in a bioreactor and to produce high yield of BmR1 recombinant antigen. Cells were grown at a controlled specific growth rate (mu(set)) during pre-induction, followed by constant feeding postinduction. The highest biomass (24.3 g l(-1)) was obtained during fed-batch process operated at mu(set) of 0.15 h(-1), whereby lower mu(set) (0.075 h(-1)) gave the highest protein production (9.82 mg l(-1)). The yield of BmR1 was increased by 1.2-fold upon induction with 1 mmol l(-1) IPTG (isopropyl-beta-d-thiogalactoside) compared to using 5 mmol l(-1) and showed a further 3.5-fold increase when the culture was induced twice at the late log phase. Combination of feeding at a lower mu(set) and twice induction with 1 mmol l(-1) IPTG yielded the best result of all variables tested, promising an improved method for BmR1 production. This method can be used to increase the production scale of the BmR1 recombinant antigen to meet the increasing demand for Brugia Rapid(), a commercial diagnostic test for detection of brugian filariasis.

Facile, reagentless and in situ release of Escherichia coli intracellular enzymes by heat-inducible autolytic vector for high-throughput screening

In an effect to broaden the application of the heat-inducible autolytic vector pUC18-cI857/p(R)-SRRz-rrnB previously developed, a new vector pUC18-cI857/p(R)(T41C)-SRRz-rrnB (pEAS-1b) was quantitatively characterized under various growth temperatures, heat induction temperatures and durations, and IPTG (isopropyl beta-d-thiogalactoside) induction times, after resolving its erratic lysis profile found previously. Escherichia coli BL21 cells harboring this vector grew well at temperatures <36 degrees C, and lysed efficiently (97.0 +/- 0.8%) just 0.5 h after heat induction at 42 degrees C for 30 min when cell growth was performed at 35 degrees C. Application of this autolytic vector either in 96-well plates, or on nitrocellulose membranes, or on agar plates led to facile, efficient and consistent release of intracellular recombinant enzymes (e.g., a lysis efficiency of 91.8 +/- 1.1% was obtained in 96-well plates). Further application in directed evolution was illustrated by improving the thermostability of amadoriase using this vector. This reagentless and in situ cell lysis method has the potentials for lysis of miniaturized samples in clinical diagnosis and bioanalytical detection, and even for lysis of cells in the microarray format.

Prokaryotic expression, polyclonal antibody preparation of the stilbene synthase gene from grape berry and its different expression in fruit development and under heat acclimation

Stilbene synthase (STS, EC 2.3.1.95) leads to the production of resveratrol compounds, which are major components of the phytoalexin response against fungal pathogens of the plant and are highly bioactive substances of pharmaceutical interest. STS expression and regulation are important. Temperature is one of the main external factors affecting phytoalexin accumulation in plant tissues, the effect of temperature on resveratrol synthesis and stilbene synthase expression in grape berries has not been reported before. Here we cloned the full-length sts cDNA with 1179bp from grape berry via PCR, and then introduced into an expressed plasmid pET-30a(+) vector at the EcoRI and XhoI restriction sites. With the isopropyl-beta-d-thiogalactoside (IPTG) induced, the pET-sts was highly expressed in Escherichia coli BL21 (DE3) pLysS cells. A fusion protein with the His-Tag was purified by Ni-NTA His.Bind Resin and then used as the antigen to immunize a New Zealand rabbit. Furthermore, the antiserum was precipitated by 50% saturated ammonium sulfate and DEAE-Sephadex A-50 chromatography to obtain the immunoglobulin G (IgG) fraction. These results provide a substantial basis for the further studies of the STS in grape berry as well as in other species of plants. The sts expression in fruit development and in response to heat acclimation was then assayed. The results indicated STS was regulated in fruits depending on the developmental stage and significantly accumulation of STS mRNA and synthesis of new STS protein during the early of heat acclimation, this work offers an important basis for further investigating the mechanism of post-harvest fruit adaptation to environmental stresses.

Biosynthesis of enantiopure (S)-3-hydroxybutyric acid in metabolically engineered Escherichia coli

A biosynthetic pathway for the production of (S)-3-hydroxybutyric acid (S3HB) from glucose was established in recombinant Escherichia coli by introducing the beta-ketothiolase gene from Ralstonia eutropha H16, the (S)-3-hydroxybutyryl-CoA dehydrogenase gene from R. eutropha H16, or Clostridium acetobutylicum ATCC824, and the 3-hydroxyisobutyryl-CoA hydrolase gene from Bacillus cereus ATCC14579. Artificial operon consisting of these genes was constructed and was expressed in E. coli BL21 (DE3) codon plus under T7 promoter by isopropyl beta-D: -thiogalactoside (IPTG) induction. Recombinant E. coli BL21 (DE3) codon plus expressing the beta-ketothiolase gene, the (S)-3-hydroxybutyryl-CoA dehydrogenase gene, and the 3-hydroxyisobutyryl-CoA hydrolase gene could synthesize enantiomerically pure S3HB to the concentration of 0.61 g l(-1) from 20 g l(-1) of glucose in Luria-Bertani medium. Fed-batch cultures of recombinant E. coli BL21 (DE3) codon plus were carried out to achieve higher titer of S3HB with varying induction time and glucose concentration during fermentation. Protein expression was induced by addition of 1 mM IPTG when cell concentration reached 10 and 20 g l(-1) (OD(600) = 30 and 60), respectively. When protein expression was induced at 60 of OD(600) and glucose was fed to the concentration of 15 g l(-1), 10.3 g l(-1) of S3HB was obtained in 38 h with the S3HB productivity of 0.21 g l(-1)h(-1). Lowering glucose concentration to 5 g l(-1) and induction of protein expression at 30 of OD(600) significantly reduced final S3HB concentration to 3.7 g l(-1), which also resulted in the decrease of the S3HB productivity to 0.05 g l(-1)h(-1).

Expression of soluble, biologically active recombinant human tumstatin in Escherichia coli

Tumstatin, a 28-kDa C-terminal fragment of collagen IV, is a potent anti-angiogenic protein and inhibitor of tumour growth. Recombinant tumstatin was prepared from Escherichia coli deposited as insoluble, inactive inclusion bodies. In the present study, we produced soluble and biologically active recombinant human tumstatin in E. coli by the coding region of tumstatin being linked to the 3'-end of the maltose-binding protein (MBP) gene. The fusion protein was expressed as the soluble form after induction by isopropylthio-beta-D-galactoside (IPTG). MBP-tumstatin was purified by amylose affinity chromatography. MBP can be removed by digestion with factor Xa. Expression could represent 20% of the total soluble protein in E. coli, allowing approximately 8.6 mg of highly purified protein to be obtained per litre of bacterial culture. The purified tumstatin specifically inhibited the proliferation of endothelial cells in a dose-dependent manner. Annexin V-FITC apoptotic assay showed that recombinant tumstatin induced significant increase of apoptotic endothelial cells after 20 h of exposure to 20 microg/ml tumstatin, and when tumstatin was incubated on the chicken embryo, chorioallantoic membrane at doses of 1-15 microg, there was a dramatic decrease in the microvasculature allantoids of chicken embryos neovascular vessel test in vivo demonstrated that tumstatin treatment at doses of 1-15 microg gives rise to dramatically decrease the number of neovascular vessel. Our study provides a feasible and convenient approach to produce soluble and biologically active tumstatin.

Expression and purification of Zantedeschia aethiopica agglutinin in Escherichia coli

Recombinant Zantedeschia aethiopica agglutinin (ZAA) was expressed in Escherichia coli as N-terminal His-tagged fusion. After induction with isopropylthio-beta-D-galactoside (IPTG), the recombinant ZAA was purified by metal-affinity chromatography. The purified ZAA protein was applied in anti-fungal assay and the result showed that recombinant ZAA had anti-fungal activity towards leaf mold (Fulvia fulva), one of the most serious phytopathogenic fungi causing significant yield loss of crops. This study suggests that ZAA could be an effective candidate in genetic engineering of plants for the control of leaf mold.

Production of recombinant mink growth hormone in E. coli

Escherichia coli cells expressing mink (Mustela vison) growth hormone were grown in a batch fermentation process. The expression level was estimated to be 27% of the total cellular protein after 3 h of induction with 1 mM isopropyl beta-D-thiogalactoside (IPTG). If the expression of mink growth hormone (mGH) was induced with 0.2 mM IPTG, the concentration of target protein was slightly lower and was found to be 23% at the same time after induction. mGH expressed as inclusion bodies was solubilized in 8 M urea and renatured by dilution protocol at a protein concentration of 1.4-2.1 mg/ml in the presence of glutathione pair in a final concentration of 11.3 mM. [GSH]/[GSSG] ratio equal to 2/1 was used. Two-step purification process comprising of ion-exchange chromatography on Q-Sepharose and hydrophobic chromatography on Phenyl-Sepharose was developed. Some 25-30 mg of highly purified and biologically active mGH was obtained from 4 g of biomass. The method presented in this study allows producing large quantities of mGH and considering initiation of scientific investigation on mGH effect on mink in vivo and availability in fur industry.