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1.  Activation of spleen cells by ArtinM may account for its immunomodulatory properties 
Cell and Tissue Research  2014;357(3):719-730.
ArtinM is a D-mannose-binding lectin extracted from Artocarpus heterophyllus that promotes interleukin-12 production by macrophages and dendritic cells. This property is considered responsible for T helper 1 immunity induced in vivo after ArtinM administration. In this study, we investigated the effect of native (jArtinM) and recombinant (rArtinM) forms of lectin on murine spleen cells and isolated T lymphocytes. We found that ArtinM binds to the surface of spleen cells. This interaction, which was blocked by D-mannose, induced cell activation, as manifested by increased mitochondrial activity, interleukin-2 production, and cell proliferation. We verified that a 30-times higher concentration of rArtinM was required to trigger optimal activation of spleen cells compared with that needed with jArtinM, although these proteins have identical sugar recognition properties and use the same signaling molecules to trigger cell activation. Because the distinction between native and recombinant is restricted to their tertiary structure (tetrameric and monomeric, respectively), we postulated that the multi-valence of jArtinM accounts for its superiority in promoting clustering of cell surface glycoreceptors and activation. The jArtinM and rArtinM activation effect exerted on spleen cells was reproduced on purified CD4+ T cells. Our results suggest that ArtinM interaction with T cells leads to responses that may act in concert with the interleukin-12 produced by antigen-presenting cells to modulate immunity toward the T helper 1 axis. Further studies are necessary to dissect ArtinM/T-cell interactions to more fully understand the immunomodulation induced by carbohydrate recognition.
PMCID: PMC4148593  PMID: 24842046
ArtinM; Spleen cells; T lymphocytes; Carbohydrate recognition; Immunomodulation
2.  The immunomodulatory effect of plant lectins: a review with emphasis on ArtinM properties 
Glycoconjugate Journal  2013;30(7):641-657.
Advances in the glycobiology and immunology fields have provided many insights into the role of carbohydrate-protein interactions in the immune system. We aim to present a comprehensive review of the effects that some plant lectins exert as immunomodulatory agents, showing that they are able to positively modify the immune response to certain pathological conditions, such as cancer and infections. The present review comprises four main themes: (1) an overview of plant lectins that exert immunomodulatory effects and the mechanisms accounting for these activities; (2) general characteristics of the immunomodulatory lectin ArtinM from the seeds of Artocarpus heterophyllus; (3) activation of innate immunity cells by ArtinM and consequent induction of Th1 immunity; (4) resistance conferred by ArtinM administration in infections with intracellular pathogens, such as Leishmania (Leishmania) major, Leishmania (Leishmania) amazonensis, and Paracoccidioides brasiliensis. We believe that this review will be a valuable resource for more studies in this relatively neglected area of research, which has the potential to reveal carbohydrate targets for novel prophylactic and therapeutic strategies.
PMCID: PMC3769584  PMID: 23299509
Plant lectins; ArtinM lectin; Immunomodulation; Toll-like receptor; Leishmania; Paracoccidioides brasiliensis
3.  The Recognition of N-Glycans by the Lectin ArtinM Mediates Cell Death of a Human Myeloid Leukemia Cell Line 
PLoS ONE  2011;6(11):e27892.
ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus (jackfruit), interacts with N-glycosylated receptors on the surface of several cells of hematopoietic origin, triggering cell migration, degranulation, and cytokine release. Because malignant transformation is often associated with altered expression of cell surface glycans, we evaluated the interaction of ArtinM with human myelocytic leukemia cells and investigated cellular responses to lectin binding. The intensity of ArtinM binding varied across 3 leukemia cell lines: NB4>K562>U937. The binding, which was directly related to cell growth suppression, was inhibited in the presence of Manα1-3(Manα1-6)Manβ1, and was reverted in underglycosylated NB4 cells. ArtinM interaction with NB4 cells induced cell death (IC50 = 10 µg/mL), as indicated by cell surface exposure of phosphatidylserine and disruption of mitochondrial membrane potential unassociated with caspase activation or DNA fragmentation. Moreover, ArtinM treatment of NB4 cells strongly induced reactive oxygen species generation and autophagy, as indicated by the detection of acidic vesicular organelles in the treated cells. NB4 cell death was attributed to ArtinM recognition of the trimannosyl core of N-glycans containing a ß1,6-GlcNAc branch linked to α1,6-mannose. This modification correlated with higher levels of N-acetylglucosaminyltransferase V transcripts in NB4 cells than in K562 or U937 cells. Our results provide new insights into the potential of N-glycans containing a β1,6-GlcNAc branch linked to α1,6-mannose as a novel target for anti-leukemia treatment.
PMCID: PMC3223207  PMID: 22132163
4.  Recognition of TLR2 N-Glycans: Critical Role in ArtinM Immunomodulatory Activity 
PLoS ONE  2014;9(6):e98512.
TLR2 plays a critical role in the protection against Paracoccidioides brasiliensis conferred by ArtinM administration. ArtinM, a D-mannose-binding lectin from Artocarpus heterophyllus, induces IL-12 production in macrophages and dendritic cells, which accounts for the T helper1 immunity that results from ArtinM administration. We examined the direct interaction of ArtinM with TLR2using HEK293A cells transfected with TLR2, alone or in combination with TLR1 or TLR6, together with accessory proteins. Stimulation with ArtinM induced NF-κB activation and interleukin (IL)-8 production in cells transfected with TLR2, TLR2/1, or TLR2/6. Murine macrophages that were stimulated with ArtinM had augmented TLR2 mRNA expression. Furthermore, pre-incubation of unstimulated macrophages with an anti-TLR2 antibody reduced the cell labeling with ArtinM. In addition, a microplate assay revealed that ArtinM bound to TLR2 molecules that had been captured by specific antibodies from a macrophages lysate. Notably,ArtinM binding to TLR2 was selectively inhibited when the lectin was pre-incubated with mannotriose. The biological relevance of the direct interaction of ArtinM with TLR2 glycans was assessed using macrophages from TLR2-KOmice, which produced significantly lower levels of IL-12 and IL-10 in response to ArtinM than macrophages from wild-type mice. Pre-treatment of murine macrophages with pharmacological inhibitors of signaling molecules demonstrated the involvement of p38 MAPK and JNK in the IL-12 production induced by ArtinM and the involvement ofPI3K in IL-10 production. Thus, ArtinM interacts directly with TLR2 or TLR2 heterodimers in a carbohydrate recognition-dependent manner and functions as a TLR2 agonist with immunomodulatory properties.
PMCID: PMC4043963  PMID: 24892697
5.  ArtinM offers new perspectives in the development of antifungal therapy 
The thermally dimorphic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis (PCM), the most frequent systemic mycosis that affects the rural populations in Latin America. Despite significant developments in antifungal chemotherapy, its efficacy remains limited since drug therapy is prolonged and associated with toxic side effects and relapses. In response to these challenges, it is now recognized that several aspects of antifungal immunity can be modulated to better deal with fungal infections. A common idea for halting fungal infections has been the need to activate a cell-based, pro-inflammatory Th1 immune response to improve the fungal elimination. ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has the property of modulating immunity against several intracellular pathogens. Here, we review the immunomodulatory activity of ArtinM during experimental PCM in mice. Both prophylactic and therapeutic protocols of ArtinM administration promotes a Th1 immune response balanced by IL-10, which outstandingly reduces the fungal load in organs of the treated mice while maintaining a controlled inflammation at the site of infection. A carbohydrate recognition-based interaction of ArtinM with Toll-like receptor 2 (TLR2) accounts for initiating the immunomodulatory effect of the lectin. The precise identification of the TLR2 N-glycan(s) targeted by ArtinM may support novel basis for the development of antifungal therapy.
PMCID: PMC3375580  PMID: 22715337
Paracoccidioides brasiliensis; ArtinM; immunomodulation
6.  The Lectin ArtinM Induces Recruitment of Rat Mast Cells from the Bone Marrow to the Peritoneal Cavity 
PLoS ONE  2010;5(3):e9776.
The D-mannose binding lectin ArtinM is known to recruit neutrophils, to degranulate mast cells and may have potential therapeutic applications. However, the effect of ArtinM on mast cell recruitment has not been investigated.
Male Wistar rats were injected i.p. with ArtinM or ConA (control). The ability of the lectin to degranulate peritoneal and mesenteric mast cells was examined. Recruitment of mast cells to the peritoneal cavity and mesentery after ArtinM injection was examined with or without depletion of peritoneal mast cells by distilled water.
ArtinM degranulated both peritoneal and mesentery mast cells in vitro. Three days after i.p. injection of the lectin there were reduced numbers of mast cells in the peritoneal lavage, while at 7 days post injection of ArtinM, the number of peritoneal mast cells was close to control values. Since immature mast cells are recruited from the bone marrow, the effect of the lectin on bone marrow mast cells was examined. Injection of ArtinM resulted in an increased number of mast cells in the bone marrow. To determine if degranulation of mast cells in the peritoneal cavity was required for the increase in bone marrow mast cells, the peritoneal cavity was depleted of mast cells with ultrapure water. Exposure to ArtinM increased the number of mast cells in the bone marrow of rats depleted of peritoneal mast cells.
The ArtinM induced recruitment of mast cells from the bone marrow to the peritoneal cavity may partially explain the therapeutic actions of ArtinM.
PMCID: PMC2842300  PMID: 20339538
7.  Evaluating the Equilibrium Association Constant between ArtinM Lectin and Myeloid Leukemia Cells by Impedimetric and Piezoelectric Label Free Approaches 
Biosensors  2014;4(4):358-369.
Label-free methods for evaluating lectin–cell binding have been developed to determine the lectin–carbohydrate interactions in the context of cell-surface oligosaccharides. In the present study, mass loading and electrochemical transducer signals were compared to characterize the interaction between lectin and cellular membranes by measuring the equilibrium association constant, Ka, between ArtinM lectin and the carbohydrate sites of NB4 leukemia cells. By functionalizing sensor interfaces with ArtinM, it was possible to determine Ka over a range of leukemia cell concentrations to construct analytical curves from impedimetric and/or mass-associated frequency shifts with analytical signals following a Langmuir pattern. Using the Langmuir isotherm-binding model, the Ka obtained were (8.9 ± 1.0) × 10−5 mL/cell and (1.05 ± 0.09) × 10−6 mL/cell with the electrochemical impedance spectroscopy (EIS) and quartz crystal microbalance (QCM) methods, respectively. The observed differences were attributed to the intrinsic characteristic sensitivity of each method in following Langmuir isotherm premises.
PMCID: PMC4287707  PMID: 25587428
ArtinM; lectin; myeloid leukemia cells; electrochemical impedance spectroscopy; quartz crystal microbalance; Langmuir isotherm; equilibrium association constant (Ka)
8.  Optimized Condition for Enhanced Soluble-Expression of Recombinant Mutant Anabaena Variabilis Phenylalanine Ammonia Lyase 
Advanced Pharmaceutical Bulletin  2014;4(3):261-266.
Purpose: Recently discovered Anabaena variabilis phenylalanine ammonia lyase (AvPAL) proved to be a good candidate for enzyme replacement therapy of phenylketonuria. Outstanding stability properties of a mutant version of this enzyme, produced already in our laboratory, have led us to the idea of culture conditions optimization for soluble expression of this therapeutically valuable enzyme in E. coli.
Methods: In the present study, the gene encoding mutant version of AvPAL was cloned into the pET28a expression vector. Different concentrations of IPTG, induction period, growth temperature, shaking speed, as well as different types of culture media were examined with respect to the amount of recombinant protein produced and specific activity of the enzyme.
Results: Based upon our findings, maximum amount of active mutant enzyme was attained by addition of 0.5 mM IPTG at 150 rpm to the TB culture media. The yield of active enzyme at cluture tempreature of 25 °C and induction period of 18 hour was the highest.
Conclusion: The results of this study indicated that the yield of mutant AvPAL production in E. coli can be affected mainly by culture temperature and inducer concentration.
PMCID: PMC3992962  PMID: 24754010
Phenylalanine ammonia lyase; Soluble expression; Optimization; Specific activity
9.  Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation 
Silk-elastin-like proteins (SELPs) combining the physicochemical and biological properties of silk and elastin have a high potential for use in the pharmaceutical, regenerative medicine and materials fields. Their development for use is however restrained by their production levels. Here we describe the batch production optimisation for a novel recently described SELP in the pET-E. coli BL21(DE3) expression system. Both a comprehensive empirical approach examining all process variables (media, induction time and period, temperature, pH, aeration and agitation) and a detailed characterisation of the bioprocess were carried out in an attempt to maximise production with this system.
This study shows that maximum SELP volumetric production is achieved at 37°C using terrific broth at pH 6–7.5, a shake flask volume to medium volume ratio of 10:1 and an agitation speed of 200 rpm. Maximum induction is attained at the beginning of the stationary phase with 0.5 mM IPTG and an induction period of at least 4 hours. We show that the selection agents ampicillin and carbenicillin are rapidly degraded early in the cultivation and that plasmid stability decreases dramatically on induction. Furthermore, acetate accumulates during the bioprocess to levels which are shown to be inhibitory to the host cells. Using our optimised conditions, 500 mg/L of purified SELP was obtained.
We have identified the optimal conditions for the shake flask production of a novel SELP with the final production levels obtained being the highest reported to date. While this study is focused on SELPs, we believe that it could also be of general interest to any study where the pET (ampicillin selective marker)-E. coli BL21(DE3) expression system is used. In particular, we show that induction time is critical in this system with, in contrast to that which is generally believed, optimal production being obtained by induction at the beginning of the stationary phase. Furthermore, we believe that we are at or near the maximum productivity for the system used, with rapid degradation of the selective agent by plasmid encoded β-lactamase, plasmid instability on induction and high acetate production levels being the principal limiting factors for further improved production.
PMCID: PMC3623843  PMID: 23446293
Biopolymers; Silk-elastin like polymers; pET-E. coli BL21(DE3); Batch production
10.  Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain 
The expression of heterologous proteins in Escherichia coli is strongly affected by codon bias. This phenomenon occurs when the codon usage of the mRNA coding for the foreign protein differs from that of the bacterium. The ribosome pauses upon encountering a rare codon and may detach from the mRNA, thereby the yield of protein expression is reduced. Several bacterial strains have been engineered to overcome this effect. However, the increased rate of translation may lead to protein misfolding and insolubilization. In order to prove this assumption, the solubility of several recombinant proteins from plants was studied in a codon bias-adjusted E. coli strain.
The expression of eight plant proteins in Escherichia coli BL21(DE3)-pLysS and BL21(DE3)-CodonPlus-pRIL was systematically studied. The CodonPlus strain contains extra copies of the argU, ileY, and leuW tRNA genes, which encode tRNAs that recognize the codons AGA/AGG, AUA and CUA, respectively (RIL codons). The level of expression and solubility of the recombinant proteins were analyzed by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting. We found that for all proteins the solubility was at least 25% in the BL21(DE3)-pLysS strain. However, when expressed in the BL21(DE3)-CodonPlus-pRIL strain, proteins having more than 5% of amino acids coded by RIL codons were localized mainly in the insoluble fraction. Also, their expression caused retarded growth and low cell yield in the codon bias-adjusted strain at all temperatures tested. On the contrary, the solubility of proteins containing less than 5% of amino acids coded by RIL codons remained unchanged in both strains and their expression caused no effect on cell growth.
Our results show that the expression of heterologous proteins coded by high RIL codon content coding sequences in a codon bias-adjusted strain is detrimental for their solubility. Our data support the hypothesis that the possible elimination of translational pauses that increase translation rate leads to protein misfolding and aggregation. This stresses the importance of strain selection according to codon content in any scheme where a large amount of biologically active product is desirable.
PMCID: PMC2723077  PMID: 19630980
11.  Optimization of the expression of reteplase in Escherichia coli 
Reteplase is a segment of tissue plasminogen activator used for the removal of thrombi in blood vessels. In the present study the cloned reteplase gene was used for its expression in competent E. coli. The recombinant plasmid, pET15b/reteplase (rpET-BL21), was transformed into competent E. coli strain BL21 (DE3) cells. Overnight culture of the transformed bacteria was induced by the addition of isopropylthio-ß-Dgalactoside (IPTG) to the final concentrations of 0.25, 0.5, 1 and 1.5 mM. Also, the effects of different temperatures(25, 30, 37 and 39°C), shaking speeds (100, 170 and 190 rpm), and various glucose concentrations (0.25, 0.5, 0.75 and 1 mM) on the expression of reteplase were examined. Samples were analyzed by SDS-PAGE. Maximum amount of protein production was obtained by the addition of 1 mM IPTG at 37°C, 100 rpm of shaking speed in the absence of glucose.
PMCID: PMC3249778  PMID: 22224091
Reteplase; Optimization; Expression; Temperature; Shaking speed; Glucose
12.  Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor 
BMC Research Notes  2014;7(1):671.
Leptospirosis is a zoonose that is increasingly endemic in built-up areas, especially where there are communities living in precarious housing with poor or non-existent sanitation infrastructure. Leptospirosis can kill, for its symptoms are easily confused with those of other diseases. As such, a rapid diagnosis is required so it can be treated effectively. A test for leptospirosis diagnosis using Leptospira Immunoglobulin-like (Lig) proteins is currently at final validation at Fiocruz.
In this work, the process for expression of LigB (131-645aa) in E. coli BL21 (DE3)Star™/pAE was evaluated. No significant difference was found for the experiments at two different pre-induction temperatures (28°C and 37°C). Then, the strain was cultivated at 37°C until IPTG addition, followed by induction at 28°C, thereby reducing the overall process time. Under this condition, expression was assessed using central composite design for two variables: cell growth at which LigB (131-645aa) was induced (absorbance at 600 nm between 0.75 and 2.0) and inducer concentration (0.1 mM to 1 mM IPTG). Both variables influenced cell growth and protein expression. Induction at the final exponential growth phase in shaking flasks with Absind  = 2.0 yielded higher cell concentrations and LigB (131-645aa) productivities. IPTG concentration had a negative effect and could be ten-fold lower than the concentration commonly used in molecular biology (1 mM), while keeping expression at similar levels and inducing less damage to cell growth. The expression of LigB (131-645aa) was associated with cell growth. The induction at the end of the exponential phase using 0.1 mM IPTG at 28°C for 4 h was also performed in microbioreactors, reaching higher cell densities and 970 mg/L protein. LigB (131-645aa) was purified by nickel affinity chromatography with 91% homogeneity.
It was possible to assess the effects and interactions of the induction variables on the expression of soluble LigB (131-645aa) using experimental design, with a view to improving process productivity and reducing the production costs of a rapid test for leptospirosis diagnosis.
PMCID: PMC4190419  PMID: 25252618
Leptospira; Leptospirosis; Diagnosis; Statistical experimental design; Microbioreactor
13.  Cloning of a novel xylanase gene from a newly isolated Fusarium sp. Q7-31 and its expression in Escherichia coli 
Brazilian Journal of Microbiology  2012;43(1):405-417.
A strain of Q7–31 was isolated from Qinghai-Tibet Plateau and was identified as Fusarium sp. based on its morphological characteristics and ITS rDNA gene sequence analysis. It has the highest capacity of degrading cell wall activity compared with other 11 strains. To do research on its xylanase activity of Fusarium sp. Q7–31 while the degrading the rice cell walls, the complete gene xyn8 that encodes endo-1, 4-β-xylanase secreted by Fusarium sp. Q7–31 was cloned and sequenced. The coding region of the gene is separated by two introns of 56bp and 55bp. It encodes 230 amino acid residues of a protein with a calculated molecular weight of 25.7 kDa. The animo acids sequence of xyn8 gene has higher similarity with those of family 11 of glycosyl hydrolases reported from other microorganisms. The nature peptide encodeing cDNA was subcloned into pGEX5x-1 expression vector. The recombinant plasmid was expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL, and xylanase activity was measured. The expression fusion protein was identified by SDS-PAGE and Western blotting, a new specific band of about 52kDa was identified when induced by IPTG. Enzyme activity assay verified the recombinants proteins as a xylanase. A maxium activity of 2.34U/ mg, the xylanase had optimal activity at pH 6.0 and temperature 40℃.
PMCID: PMC3768979  PMID: 24031846
xylanase; Gene cloning; Fusarium sp.
14.  Overexpression of Recombinant Human Beta Interferon (rhINF-β) in Periplasmic Space of Escherichia coli  
Human Interferon β (INF-β) is a member of cytokines family which different studies have shown its immunomodulatory and antiviral activities. In this study an expression vector was designed and constructed for expression of human INF-β-1b either in shake flasks or bench top bioreactor. The designed vector was constructed based upon pET-25b(+) with T7 promoter. Recombinant human beta interferon (rhINF-β) was codon optimized and overexpressed as a soluble, N-terminal pelB fusion protein and secreted into the periplasmic space of Escherichia coli BL21 (DE3). The sugar, Isopropyl-β-D-thiogalactopyranoside (IPTG) was used as a chemical inducer for rhINF-β production in the shake flasks and bench top bioreactor. Timing of beta interferon expression was controlled by using the T7 promoter. The rhINF-β protein was extracted from periplasmic space by osmotic shock treatment and the expression of the beta interferon encoding gene in random selected transformants, was confirmed by western and dot blot methods. The maximum of product formation achieved at the OD600nm = 3.42 was found to be 35 % of the total protein content of the strain which translates to 0.32 g L-1. The constructed vector could efficiently overexpress the rhINF-β into the periplasmic space of E. coli. The obtained yield of the produced rhINF-β was more than previous reports. The system is easily adapted to include other vectors, tags or fusions and therefore has the potential to be broadly applicable to express other recombinant proteins.
PMCID: PMC3977065  PMID: 24711841
Periplasmic expression; Escherichia coli; Interferon beta; Plasmid; Expression system
15.  Utilizing high-throughput experimentation to enhance specific productivity of an E.coli T7 expression system by phosphate limitation 
BMC Biotechnology  2011;11:22.
The specific productivity of cultivation processes can be optimized, amongst others, by using genetic engineering of strains, choice of suitable host/vector systems or process optimization (e.g. choosing the right induction time). A further possibility is to reduce biomass buildup in favor of an enhanced product formation, e.g. by limiting secondary substrates in the medium, such as phosphate. However, with conventional techniques (e.g. small scale cultivations in shake flasks), it is very tedious to establish optimal conditions for cell growth and protein expression, as the start of protein expression (induction time) and the degree of phosphate limitation have to be determined in numerous concerted, manually conducted experiments.
We investigated the effect of different induction times and a concurrent phosphate limitation on the specific productivity of the T7 expression system E.coli BL21(DE3) pRhotHi-2-EcFbFP, which produces the model fluorescence protein EcFbFP upon induction. Therefore, specific online-monitoring tools for small scale cultivations (RAMOS, BioLector) as well as a novel cultivation platform (Robo-Lector) were used for rapid process optimization. The RAMOS system monitored the oxygen transfer rate in shake flasks, whereas the BioLector device allowed to monitor microbial growth and the production of EcFbFP in microtiter plates. The Robo-Lector is a combination of a BioLector and a pipetting robot and can conduct high-throughput experiments fully automated. By using these tools, it was possible to determine the optimal induction time and to increase the specific productivity for EcFbFP from 22% (for unlimited conditions) to 31% of total protein content of the E.coli cells via a phosphate limitation.
The results revealed that a phosphate limitation at the right induction time was suitable to redirect the available cellular resources during cultivation to protein expression rather than in biomass production. To our knowledge, such an effect was shown for the first time for an IPTG-inducible expression system. Finally, this finding and the utilization of the introduced high-throughput experimentation approach could help to find new targets to further enhance the production capacity of recombinant E.coli-strains.
PMCID: PMC3068942  PMID: 21414195
16.  Codon Preference Optimization Increases Heterologous PEDF Expression 
PLoS ONE  2010;5(11):e15056.
Pigment epithelium-derived factor (PEDF) is widely known for its neurotrophic and antiangiogenic functions. Efficacy studies of PEDF in animal models are limited because of poor heterologous protein yields. Here, we redesigned the human PEDF gene to preferentially match codon frequencies of E coli without altering the amino acid sequence. Following de novo synthesis, codon optimized PEDF (coPEDF) and the wtPEDF genes were cloned into pET32a containing a 5′ thioredoxin sequence (Trx) and the recombinant Trx-coPEDF or Trx-wtPEDF fusion constructs expressed in native and two tRNA augmented E coli hosts - BL21-CodonPlus(DE3)-RIL and BL21-CodonPlus(DE3)-RP, carrying extra copies of tRNAarg,ile,leu and tRNAarg,pro genes , respectively. Trx-PEDF fusion proteins were isolated using Ni-NTA metal affinity chromatography and PEDF purified after cleavage with factor Xα. Protein purity and identity were confirmed by western blot, MALDI-TOF, and UV/CD spectral analyses. Expression of the synthetic gene was ∼3.4 fold greater (212.7 mg/g; 62.1 mg/g wet cells) and purified yields ∼4 fold greater (41.1 mg/g; 11.3 mg/g wet cell) than wtPEDF in the native host. A small increase in expression of both genes was observed in hosts supplemented with rare tRNA genes compared to the native host but expression of coPEDF was ∼3 fold greater than wtPEDF in both native and codon-bias-adjusted E coli strains. ΔGs at −3 to +50 of the Trx site of both fusion genes were −3.9 kcal/mol. Functionally, coPEDF was equally as effective as wtPEDF in reducing oxidative stress, promoting neurite outgrowth, and blocking endothelial tube formation. These findings suggest that while rare tRNA augmentation and mRNA folding energies can significantly contribute to increased protein expression, preferred codon usage, in this case, is advantageous to translational efficiency of biologically active PEDF in E coli. This strategy will undoubtedly fast forward studies to validate therapeutic utility of PEDF in vivo.
PMCID: PMC2994832  PMID: 21152082
17.  Efficient Production of an Engineered Apoptin from Chicken Anemia Virus in a Recombinant E. coli for Tumor Therapeutic Applications 
BMC Biotechnology  2012;12:27.
Apoptin, a nonstructural protein encoded by the VP3 gene of chicken anemia virus (CAV), has been shown to not only induce apoptosis when introduced into the precursors of chicken thymocytes, but has been found to specifically kill human cancer cells, tumor cell and transformed cells without affecting the proliferation of normal cells. This tumor-specific apoptotic characteristic of the protein potentially may allow the development of a protein drug that has applications in tumor therapy. However, several major problems, which include poor expression and poor protein solubility, have hampered the production of apoptin in bacteria.
Significantly increased expression of recombinant full-length apoptin that originated from chicken anemia virus was demonstrated using an E. coli expression system. The CAV VP3 gene was fused with a synthetic sequence containing a trans-acting activator of transcription (TAT) protein transduction domain (PTD). The resulting construct was cloned into various different expression vectors and these were then expressed in various E. coli strains. The expression of the TAT-Apoptin in E. coli was significantly increased when TAT-Apoptin was fused with GST-tag rather than a His-tag. When the various rare amino acid codons of apoptin were optimized, the expression level of the GST-TAT-Apoptinopt in E. coli BL21(DE3) was significantly further increased. The highest protein expression level obtained was 8.33 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 4 h at 25 °C. Moreover, approximately 90% of the expressed GST-TAT-Apoptinopt under these conditions was soluble. After purification by GST affinity chromatography, the purified recombinant TAT-Apoptinopt protein was used to evaluate the recombinant protein’s apoptotic activity on tumor cells. The results demonstrated that the E. coli-expressed GST-TAT-apoptinopt showed apoptotic activity and was able to induce human premyelocytic leukemia HL-60 cells to enter apoptosis.
On expression in E. coli, purified recombinant TAT-Apoptinopt that has been fused to a GST tag and had its codons optimized, was found to have great potential. This protein may in the future allow the development of a therapeutic protein that is able to specifically kill tumor cells.
PMCID: PMC3443062  PMID: 22672291
18.  Enhancement of solubility in Escherichia coli and purification of an aminotransferase from Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B1 
Fumonisin B1 is a cancerogenic mycotoxin produced by Fusarium verticillioides and other fungi. Sphingopyxis sp. MTA144 can degrade fumonisin B1, and a key enzyme in the catabolic pathway is an aminotransferase which removes the C2-amino group from hydrolyzed fumonisin B1. In order to study this aminotransferase with respect to a possible future application in enzymatic fumonisin detoxification, we attempted expression of the corresponding fumI gene in E. coli and purification of the enzyme. Since the aminotransferase initially accumulated in inclusion bodies, we compared the effects of induction level, host strain, expression temperature, solubility enhancers and a fusion partner on enzyme solubility and activity.
When expressed from a T7 promoter at 30°C, the aminotransferase accumulated invariably in inclusion bodies in DE3 lysogens of the E. coli strains BL21, HMS174, Rosetta 2, Origami 2, or Rosetta-gami. Omission of the isopropyl-beta-D-thiogalactopyranoside (IPTG) used for induction caused a reduction of expression level, but no enhancement of solubility. Likewise, protein production but not solubility correlated with the IPTG concentration in E. coli Tuner(DE3). Addition of the solubility enhancers betaine and sorbitol or the co-enzyme pyridoxal phosphate showed no effect. Maltose-binding protein, used as an N-terminal fusion partner, promoted solubility at 30°C or less, but not at 37°C. Low enzyme activity and subsequent aggregation in the course of purification and cleavage indicated that the soluble fusion protein contained incorrectly folded aminotransferase. Expression in E. coli ArcticExpress(DE3), which co-expresses two cold-adapted chaperonins, at 11°C finally resulted in production of appreciable amounts of active enzyme. Since His tag-mediated affinity purification from this strain was hindered by co-elution of chaperonin, two steps of chromatography with optimized imidazole concentration in the binding buffer were performed to obtain 1.45 mg of apparently homogeneous aminotransferase per liter of expression culture.
We found that only reduction of temperature, but not reduction of expression level or fusion to maltose-binding protein helped to produce correctly folded, active aminotransferase FumI in E. coli. Our results may provide a starting point for soluble expression of related aminotransferases or other aggregation-prone proteins in E. coli.
PMCID: PMC2933618  PMID: 20718948
19.  Cloning and Expression of S1 Subunit of Pertussis Toxin in Escherichia coli  
Bordetella pertussis is a gram negative bacterium that causes respiratory tract infection in human (whooping cough). Pertussis toxin (PT) is the main component of current acellular pertussis vaccine and the S1 (subunit1) is the main immunogenic part of it. Thus, S1 has been the target of many studies as a potent candidate of acellular vaccine against Bordetella pertussis, lacking the side effects of whole cell based ones. S1 gene was amplified and inserted in three expression vectors including pET-14b, pET-22b(+) and pAED4. The possibility and level of expression of these constructs were investigated in BL21 (DE3) strain of Escherichia coli (E.coli) as expression host. The highest expression was in pET-22b(+)-S1. Best expression achieved 6 hr post induction with 0.2 mM IPTG in LB broth containing ampicillin, at 30°C with shaking (250 rpm). Recombinant S1 protein was observed in two distinct separated proteins with 28 and 31 kDa estimated molecular weight. In spite of toxicity of PT and S1 in the E.coli, considerable amount of S1 was expressed in E.coli. Two rS1 bands were detected on SDS-PAGE. Both were confirmed as S1 in western blot with specific monoclonal and polyclonal antibodies against pertussis toxin. Appearance of two distinct bands could be the result of leader peptidase activity or nonspecific peptidase from E.coli on recombinant S1. As the recombinant S1 is a suitable antigen for studies as a candidate acellular vaccine or development of ELISA for detection of Bordetella pertussis, further studies are underway.
PMCID: PMC3558169  PMID: 23407437
Bordetella pertussis; Escherichia coli; Pertussis toxin
20.  Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E. coli cultures 
Fab antibody fragments in E. coli are usually directed to the oxidizing periplasmic space for correct folding. From periplasm Fab fragments may further leak into extracellular medium. Information on the cultivation parameters affecting this leakage is scarce, and the unpredictable nature of Fab leakage is problematic regarding consistent product recovery. To elucidate the effects of cultivation conditions, we investigated Fab expression and accumulation into either periplasm or medium in E. coli K-12 and E. coli BL21 when grown in different types of media and under different aeration conditions.
Small-scale Fab expression demonstrated significant differences in yield and ratio of periplasmic to extracellular Fab between different culture media and host strains. Expression in a medium with fed-batch-like glucose feeding provided highest total and extracellular yields in both strains. Unexpectedly, cultivation in baffled shake flasks at 150 rpm shaking speed resulted in higher yield and accumulation of Fabs into culture medium as compared to cultivation at 250 rpm. In the fed-batch medium, extracellular fraction in E. coli K-12 increased from 2-17% of total Fab at 250 rpm up to 75% at 150 rpm. This was partly due to increased lysis, but also leakage from intact cells increased at the lower shaking speed. Total Fab yield in E. coli BL21 in glycerol-based autoinduction medium was 5 to 9-fold higher at the lower shaking speed, and the extracellular fraction increased from ≤ 10% to 20-90%. The effect of aeration on Fab localization was reproduced in multiwell plate by variation of culture volume.
Yield and leakage of Fab fragments are dependent on expression strain, culture medium, aeration rate, and the combination of these parameters. Maximum productivity in fed-batch-like conditions and in autoinduction medium is achieved under sufficiently oxygen-limited conditions, and lower aeration also promotes increased Fab accumulation into extracellular medium. These findings have practical implications for screening applications and small-scale Fab production, and highlight the importance of maintaining consistent aeration conditions during scale-up to avoid changes in product yield and localization. On the other hand, the dependency of Fab leakage on cultivation conditions provides a practical way to manipulate Fab localization.
PMCID: PMC3733871  PMID: 23895637
Fab fragment; Periplasmic expression; Oxygen transfer; Fed-batch; Autoinduction
21.  Cheese whey-induced high-cell-density production of recombinant proteins in Escherichia coli 
Use of lactose-rich concentrates from dairy processes for the induction of recombinant gene's expression has not received much attention although they are interesting low cost substrates for production of recombinant enzymes. Applicability of dairy waste for induction of recombinant genes in Escherichia coli was studied. Clones expressing Lactobacillus phage muramidase and Lactobacillus alcohol dehydrogenase were used for the experiments.
Shake flask cultivations in mineral salt medium showed that cheese whey or deproteinised whey induced gene expression as efficiently as IPTG (isopropyl-β-D-thiogalactopyranoside) or pure lactose. Addition of yeast extract or proteolytically degraded whey proteins did not improve the recombinant protein yield. In contrast, addition of yeast extract to the well-balanced mineral salt medium decreased the product yield. Feeding with glycerol provided sufficient amount of easily assimilable carbon source during the induction period without preventing lactose intake and induction by lactose. High-cell-density fed-batch cultivations showed that product yields comparable to IPTG-induction can be achieved by feeding bacteria with a mixture of glycerol and concentrated whey permeate during the induction.
Whey and concentrated whey permeate can be applied as an alternative inducer in recombinant high-cell-density fed-batch fermentations. The yield of the recombinant product was comparable to fermentations induced by IPTG. In low-cell-density shake flask experiments the yield was higher with whey or whey permeate than with IPTG.
PMCID: PMC155635  PMID: 12740045
22.  Tat Peptide-Mediated Soluble Expression of the Membrane Protein LSECtin-CRD in Escherichia coli 
PLoS ONE  2013;8(12):e83579.
The human liver and lymph node sinusoidal endothelial cell C-type lectin (hLSECtin), a type II integral membrane protein, containing a Ca2+-dependent carbohydrate recognition domain (CRD), has a well-established biological activity, yet its three-dimensional structure is unknown due to low expression yields and aggregation into inclusion bodies. Previous study has demonstrated that the HIV-1 virus-encoded Tat peptide (‘YGRKKRRQRRR’) can increase the yields and the solubility of heterologous proteins. However, whether the Tat peptide could promote the high-yield and soluble expression of membrane proteins in Escherichia coli is not known. Therefore, the prokaryotic expression vector pET28b-Tat-hLSECtin-CRD (using pET28b and pET28b-hLSECtin-CRD as controls) was constructed, and transformed into E. coli BL21 (DE3) cells and induced with isopropyl-β-d-thiogalactoside (IPTG) followed with identifying by SDS-PAGE and Western blot. Subsequently, the bacterial subcellular structure, in which overexpressed the heterologous proteins Tat-hLSECtin-CRD and Tat-free hLSECtin-CRD, was analyzed by transmission electron microscope (TEM) respectively, and the mannose-binding activity of Tat-hLSECtin-CRD was also determined. Expectedly, the solubility of Tat-LSECtin-CRD significantly increased compared to Tat-free LSECtin-CRD (**p < 0.01) with prolonged time, and the Tat-LSECtin-CRD had a significant mannose-binding activity. The subcellular structure analysis indicated that the bacterial cells overexpressed Tat-hLSECtin-CRD exhibited denser region compared with controls, while dot denser region aggregated in the two ends of bacterial cells overexpressed Tat-free hLSECtin-CRD. This study provided a novel method for improving the soluble expression of membrane proteins in prokaryotic systems by fusion with the Tat peptide, which may be potentially expanded to the expression of other membrane proteins.
PMCID: PMC3865297  PMID: 24358298
23.  Optimizing HIV-1 protease production in Escherichia coli as fusion protein 
Human immunodeficiency virus (HIV) is the etiological agent in AIDS and related diseases. The aspartyl protease encoded by the 5' portion of the pol gene is responsible for proteolytic processing of the gag-pol polyprotein precursor to yield the mature capsid protein and the reverse transcriptase and integrase enzymes. The HIV protease (HIV-1Pr) is considered an attractive target for designing inhibitors which could be used to tackle AIDS and therefore it is still the object of a number of investigations.
A recombinant human immunodeficiency virus type 1 protease (HIV-1Pr) was overexpressed in Escherichia coli cells as a fusion protein with bacterial periplasmic protein dithiol oxidase (DsbA) or glutathione S-transferase (GST), also containing a six-histidine tag sequence. Protein expression was optimized by designing a suitable HIV-1Pr cDNA (for E. coli expression and to avoid autoproteolysis) and by screening six different E. coli strains and five growth media. The best expression yields were achieved in E. coli BL21-Codon Plus(DE3)-RIL host and in TB or M9 medium to which 1% (w/v) glucose was added to minimize basal expression. Among the different parameters assayed, the presence of a buffer system (based on phosphate salts) and a growth temperature of 37°C after adding IPTG played the main role in enhancing protease expression (up to 10 mg of chimeric DsbA:HIV-1Pr/L fermentation broth). GST:HIVPr was in part (50%) produced as soluble protein while the overexpressed DsbA:HIV-1Pr chimeric protein largely accumulated in inclusion bodies as unprocessed fusion protein. A simple refolding procedure was developed on HiTrap Chelating column that yielded a refolded DsbA:HIV-1Pr with a > 80% recovery. Finally, enterokinase digestion of resolubilized DsbA:HIV-1Pr gave more than 2 mg of HIV-1Pr per liter of fermentation broth with a purity ≤ 80%, while PreScission protease cleavage of soluble GST:HIVPr yielded ~ 0.15 mg of pure HIV-1Pr per liter.
By using this optimized expression and purification procedure fairly large amounts of good-quality HIV-1Pr recombinant enzyme can be produced at the lab-scale and thus used for further biochemical studies.
PMCID: PMC3141379  PMID: 21718537
24.  Production of recombinant Entamoeba histolytica pyruvate phosphate dikinase and its application in a lateral flow dipstick test for amoebic liver abscess 
BMC Infectious Diseases  2014;14:182.
Amoebic liver abscess (ALA) is the most common clinical manifestation of extraintestinal amoebiasis especially in developing countries, causing up to 100 000 fatal cases annually. Accurate and early diagnosis is important to prevent the disease complications, however its diagnosis still poses many challenges due to the limitations of the available detection tools. Pyruvate phosphate dikinase (PPDK), an excretory-secretory protein of E. histolytica, has been reported as a potential diagnostic marker for ALA, hence it may be exploited in the development of a new test for ALA.
Recombinant PPDK (rPPDK) was expressed, purified and evaluated by Western blot. In parallel, recombinant galactose-and-N-acetyl-D-galactosamine inhibitable lectin (Gal/GalNAc lectin) was produced and tested similarly. The protein identity was confirmed by analysis using MALDI-TOF/TOF. A lateral flow dipstick (LFD) test using rPPDK was subsequently developed (rPPDK-LFD) and evaluated for serodiagnosis of ALA.
rPPDK was expressed as soluble protein after 4 hours of induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 30°C. Purification using nickel-nitrilotriacetic acid (Ni-NTA) resin yielded 1.5 mg of rPPDK from 1 L of culture with estimated molecular mass of 98 kDa on SDS-PAGE. Western blots using sera from patients with ALA, healthy individuals and other diseases probed with anti-human IgG4-HRP showed the highest sensitivity (93.3%) and specificity (100%); as compared to blots using IgG and IgG1 as secondary antibodies. Moreover, rPPDK showed better specificity when compared to rGal/GalNAc lectin. In the development of the LFD test, the optimum amount of rPPDK was 0.625 μg per dipstick and the optimum working concentration of colloidal gold conjugated anti-human IgG4 was optical density (OD) 5 (1.7 μg of anti-human IgG4). Evaluation of rPPDK-LFD using ALA patients and controls serum samples showed 87% diagnostic sensitivity and 100% specificity.
The developed rPPDK-LFD showed good potential for rapid diagnosis of ALA, and merit further multicentre validation using larger number of serum samples.
PMCID: PMC3986461  PMID: 24708664
Entamoeba histolytica; Amoebic liver abscess; Diagnosis; Recombinant pyruvate phosphate dikinase; Western blot; Lateral flow dipstick test
25.  Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli 
BMC Biotechnology  2013;13:107.
HIV genome is packaged and organized in a conical capsid, which is made up of ~1,500 copies of the viral capsid protein p24 (CA). Being a primary structural component and due to its critical roles in both late and early stages of the HIV replication cycle, CA has attracted increased interest as a drug discovery target in recent years. Drug discovery studies require large amounts of highly pure and biologically active protein. It is therefore desirable to establish a simple and reproducible process for efficient production of HIV-1 CA.
In this work, 6-His-tagged wild type CA from HIV-1 (NL4.3) was expressed in rare tRNA-supplemented NiCo21(DE3) Escherichia coli, and its production was studied in shake flask culture condition of expression. Influences of various key cultivation parameters were examined to identify optimal conditions for HIV-1 CA production. It was found that a culture temperature of 22°C and induction with 0.05 mM IPTG at the early stage of growth were ideal, leading to a maximum biomass yield when grown in Super broth supplemented with 1% glucose. With optimized culture conditions, a final biomass concentration of ~27.7 g L-1 (based on optical density) was obtained in 12 hours post-induction, leading to a yield of about ~170 mg L-1 HIV-1 CA. A two-step purification strategy (chitin beads + IMAC) was employed, which efficiently removed metal affinity resin-binding bacterial proteins that contaminate recombinant His-tagged protein preparation, and resulted in highly pure HIV-1 CA. The purified protein was capable of polymerization when tested in an in vitro polymerization assay.
By using this optimized expression and purification procedure, milligram amounts of highly pure and polymerization-competent recombinant HIV-1 CA can be produced at the lab-scale and thus used for further biochemical studies.
PMCID: PMC4235032  PMID: 24304876

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