IPTG Research Articles

Development of the Mammalian Expression Vector System that can be Induced by IPTG and/or Lactose.

Techniques used for the regulation of gene expression facilitate studies of gene function and treatment of diseases via gene therapy. Many tools have been developed for the regulation of gene expression in mammalian cells. The Lac operon system induced with isopropyl β-D-1- thiogalactopyranoside (IPTG) is one of the employed inducible systems. IPTG mimics the molecular structure of allolactose and has a strong affinity for the corresponding repressor. IPTG is known to rapidly penetrate into mammalian cells and exhibits low toxicity. In the present study, we developed a new inducible expression system that could regulate the expression of genes in mammalian cells using IPTG. Here we confirm that unlike other vector systems based on the Lac operon, this expression system allows regulation of gene expression with lactose in the mammalian cells upon transfection. The co-treatment with IPTG and lactose could improve the regulatory efficiency of the specific target gene expression. The regulation of gene expression with lactose has several benefits. Lactose is safe in humans as compared to other chemical substances and is easily available, making this technique very cost-effective.

Cloning and sequence analysis of leptin receptor overlapping transcript-like 1 gene from Dermatophagoides farinae

To obtain the leptin receptor overlapping transcript-like 1 encoding gene (LepROTL1 gene) from Dermatophagoides farina, investigate the molecular characteristics of the gene and construct a prokaryotic expression vector to express this gene. The LepROTL1 gene-encoding sequence fragments were captured based on the transcriptome sequencing results, and the full-length gene fragments were amplified from total RNA of D. farinae using a RT-PCR assay, and used to construct the expression plasmid pET28a(+)-LepROTL1, followed by sequencing. The plasmid was transformed into E. coli BL21 (DE3) T1R for the induction of IPTG expression. The expression product was characterized by SDS-PAGE and Western blotting. Bioinformatics analyses were performed to analyze the sequence and the molecular characteristics of its encoded protein. The amplification products of the RT-PCR assay showed a clear band on agarose gel electrophoresis, and sequencing analysis of the pET28a(+)-LepROTL1 plasmid showed 417 bp in length of the coding gene from the start codon ATG to the termination codon TAA. Following the plasmid transformation into E. coli and induction with IPTG, a specific band was seen on SDS-PAGE, indicating successful expression. Bioinformatics analysis showed that the LepROTL1 gene-encoded protein was composed of 134 amino acids, and had a relative molecular weight of 14 378.13 Da, a hydrophilicity index of 1.149, and certain hydrophobicity. The secondary structure was composed of alpha-helix (19 aa, 14.18%), extended strand (48 aa, 35.82%) and random coil (67 aa, 50.00%). The deduced amino acid sequence was used to obtain homologous genes by BLAST, and the phylogenetic tree showed that D. farinae was clustered with D. pteronyssinus. The full-length sequences and expression plasmid of the LepROTL1 gene are obtained, and the molecular features of the gene are demonstrated using bioinformatics analyses, which provide insights into further studies on the gene.

A protocol for recombinant protein quantification by densitometry.

The protein purity is generally checked using SDS‐PAGE, where densitometry could be used to quantify the protein bands. In literature, few studies have been reported using image analysis for the quantification of protein in SDS‐PAGE: that is, imaged with Stain‐Free™ technology. This study presents a protocol of image analysis for electrophoresis gels that allows the quantification of unknown proteins using the molecular weight markers as protein standards. Escherichia coli WK6/pHEN6 encoding the bispecific nanobody CH10‐12 engineered by the Pasteur Institute of Tunisia was cultured in a bioreactor and induced with isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) at 28°C for 12 hr. Periplasmic proteins extracted by osmotic shock were purified by immobilized metal affinity chromatography (IMAC). Images of the SDS‐PAGE gels were analyzed using ImageJ, and the lane profiles were obtained in grayscale and uncalibrated optical density. Protein load and peak area were linearly correlated, and optimal image processing was then performed by background subtraction using the rolling ball algorithm with radius size 250 pixels. No brightness and contrast adjustment was applied. The production of the nanobody CH10‐12 was obtained through a fed‐batch strategy and quantified using the band of 50 kDa in the marker as reference for 750 ng of recombinant protein. The molecular weight marker was used as a sole protein standard for protein quantification in SDS‐PAGE gel images.

Highly effective methods for expression/purification of recombinant human HSP90 and its four distinct (N‐LR‐M‐C) domains

Heat shock protein 90 (HSP90) plays essential roles in normal physiology to maintain homeostasis and in many human diseases. It comprises four distinct domains, including NH2‐terminal (N), charged linker region (LR), middle (M), and COOH‐terminal (C) domains, all of which are important for regulating HSP90 biological functions. We reported herein detailed protocols to produce recombinant full‐length (FL) and all these four domains of human HSP90 from Escherichia coli. cDNAs encoding FL, N, LR, M and C domains of human HSP90α were amplified and cloned into pET‐32b(+) expression vector. All HSP90 constructs were expressed as soluble Trx‐His‐S tagged proteins after induction with 0.25 mM isopropyl‐β‐D‐thiogalactopyranoside (IPTG) at 18°C overnight and further purified by affinity chromatography using nickel‐nitrilotriacetic acid (Ni‐NTA) resin. The enterokinase (EK) digestion was optimized for efficient cleavage of the Trx‐His‐S tag from each HSP90 construct by varying concentrations of EK (0.5–1 U) and urea (0–3 M). After rebinding to Ni‐NTA resin, each HSP90 construct was highly purified and approximately 0.1–1 mg proteins were obtained from 100 ml of bacterial culture. All the purified HSP90 constructs were successfully confirmed by tandem mass spectrometry (nanoLC‐ESI‐ETD MS/MS). Our expression and purification protocols would facilitate further structural and functional studies of human HSP90.Support or Funding InformationThis study was supported by Mahidol University research grant and the Thailand Research Fund (IRN60W0004).

Prokaryotic expression, purification and identification of the N protein of middle east respiratory syndrome coronavirus

Objective To express the N protein of middle east respiratory syndrome coronavirus （ MERS-CoV） in prokaryotic expression system and evaluate the immunogenicity of the purified recombinant N protein.Methods The gene encoding N protein of MERS-CoV was synthesized and cloned into the vector pET30a to construct the recombinant expression plasmid pET30a-MERS-CoV-N.The transformed E.coli BL21 （ DE3） strains carrying expression plasmid were induced by IPTG to express N protein.The expressed protein was purified by using affinity chromatography.SDS-PAGE and Western blot assays were used to iden-tify the expressed N protein and evaluate its immunogenicity.Results The recombinant N protein was suc-cessfully expressed in soluble form with the size of 46×103 .The results of Western blot assay showed that the recombinant N protein could specifically react with rabbit serum samples positive for antibodies against N protein B-cell epitope peptide and mouse anti-His tag antibodies.No positive band against N protein was found when primary antibody was used with thirty adult serum samples from Beijing in 2008.Conclusion N protein of MERS-CoV was successfully expressed in prokaryotic expression system.The successful expres-sion of N protein laid the foundation for immunological diagnosis of N protein of MERS-CoV and researches on its immunogenicity. Key words: Middle east respiratory syndrome coronavirus; N protein; Prokaryotic expression; Purification ; Identification

Expression and antibody preparation of recombinant truncated glycoprotein of Guertu virus

Objective To express truncated glycoprotein (Gn, Gn1, Gn2, Gn3, Gc1 and Gc2) of Guertu virus (GTV) in Escherichia coli (E.coli) cells, and prepare polyclonal antibodies against recombinant proteins Gn-His, Gc1-His and Gc2-His after purification. Methods Gene fragments encoding Gn, Gn1, Gn2, Gn3, Gc1 and Gc2 of GTV DXM strain were amplified by RT-PCR, and cloned into the prokaryotic expression vector pET-32a (+ ) to construct recombinant expression plasmids. The transformed E. coli BL21(DE3) strains carrying expression plasmids were induced by IPTG to express target proteins, which were identified by SDS-PAGE. Recombinant proteins Gn-His, Gc1-His and Gc2-His were purified by nickel affinity chromatography and detected by Western blot using GTV-positive sheep serum for analysis of their antigenicity. New Zealand white rabbits were immunized with the purified recombinant proteins. The titers and specificity of serum antibodies were analyzed by ELISA. Meanwhile, eukaryotic expression vectors pcDNA3.1-Gn, pcDNA3.1-Gc1/Gc2 were constructed and transfected into mammalian Vero cells to evaluate the binding activity of rabbit polyclonal antibodies by indirect immunofluorescence method. The specific reactivity of serum antibodies to recombinant proteins was detected by Western blot. Results Restriction enzyme analysis and DNA sequencing confirmed that the recombinant expression vectors of pET-32a-Gn, pET-32a-Gn1/Gn2/Gn3, pET-32a-Gc1/Gc2, pcDNA3.1-Gn and pcDNA3.1-Gc1/Gc2 were constructed successfully. The relative molecular mass (Mr) of the expressed recombinant proteins Gn-His, Gn1/Gn2/Gn3-His, Gc1/Gc2-His were approximately 63.4×103, 37.1×103, 31.9×103, 30.8×103, 40×103 and 54.4×103, respectively. The recombinant proteins could be recognized by GTV-positive sheep serum. The titers of polyclonal antibodies against GTV Gn, Gc1 and Gc2 were 1∶409 600, 1∶204 800 and 1∶6 400, respectively. Indirect immunofluorescence assay and Western blot showed that the prepared rabbit polyclonal antibodies could specifically react with the proteins expressed in eukaryotic cells and the recombinant proteins. Conclusions The recombinant GTV glycoproteins Gn-His and Gc1/Gc2-His were efficiently expressed and purified and characterized with good immunity. The prepared polyclonal antibodies had high titers and good specificity. This study provided reference for further studying the biological function and detection methods of GTV glycoproteins and research on vaccines. Key words: Guertu Virus; Glycoprotein; Prokaryotic expression; Eukaryotic expression; Rabbit polyclonal antibody

A novel approach for high-level expression and purification of GST-fused highly thermostable Taq DNA polymerase in Escherichia coli

Polymerases are enzymes that synthesize long chains or polymers of nucleic acids including DNA or RNA from nucleotides. They assemble nucleic acids by copying a DNA or RNA template strand using base-pairing interactions. One of the polymerase enzymes, Taq DNA polymerase, originally isolated from Thermus aquaticus (Taq) is a widely used enzyme in molecular biology so far. The thermostable properties of this enzyme have contributed majorly to the specificity, automation, and efficacy of the polymerase chain reaction (PCR), making it a powerful tool for today's molecular biology researches across the globe. The purification of Taq DNA polymerase from the native host results in low yield, more labor and time consumption. Therefore, many studies have been previously conducted to obtain this enzyme using alternative hosts. So far, all the existing methodologies are more laborious, time-consuming and require heavy expense. We used a novel approach to purify the enzyme with relatively high efficiency, yield and minimum time consumption using Escherichia coli (E. coli) as an alternative host. We cloned a 2500 base pair Taq DNA polymerase gene into pGEX-4T-1 vector, containing a GST-tag, downstream of tac promoter and overexpressed it using isopropyl β-d-1-thiogalactopyranoside (IPTG) as an inducer. The enzyme was efficiently purified using novel chromatography approaches and was used in routine PCR assays in our laboratory. Our findings suggest a novel approach to facilitate the availability of polymerases for molecular and diagnostic studies. In the future, it may be used for the purification of other recombinant peptides or proteins used in structural biology and proteomics-based researches.

Homing endonuclease I-SceI-mediated Corynebacterium glutamicum ATCC 13032 genome engineering.

Corynebacterium glutamicum is widely used to produce amino acids and is a chassis for the production of value-added compounds. Effective genome engineering methods are crucial to metabolic engineering and synthetic biology studies of C. glutamicum. Herein, a homing endonuclease I-SceI-mediated genome engineering strategy was established for the model strain C. glutamicum ATCC 13032. A vegetative R6K replicon-based, suicide plasmid was employed. The plasmid, pLS3661, contains both tightly regulated, IPTG (isopropyl-β-D-1-thiogalactopyranoside)-inducible I-SceI expression elements and two I-SceI recognition sites. Following cloning of the homologous arms into pLS3661 and transfer the recombinant vector into C. glutamicum ATCC 13032, through the homologous recombination between the cloned fragment and its chromosomal allele, a merodiploid was selected under kanamycin selection. Subsequently, a merodiploid was resolved by double-stranded break repair stimulated by IPTG-stimulated I-SceI expression, generating desired mutants. The protocol obviates a pre-generated strain, transfer of a second I-SceI expression plasmid, and there is not any strain, medium, and temperature restrictions. We validated the approach via deletions of five genes (up to ~ 13.0kb) and knock-in of one DNA fragment. Furthermore, through kanamycin resistance repair, the ssDNA recombineering parameters were optimized. We hope the highly efficient method will be helpful for the studies of C. glutamicum, and potentially, to other bacteria. KEY POINTS: • Counterselection marker I-SceI-mediated C. glutamicum genome engineering • A suicide vector contains I-SceI expression elements and its recognition sites • Gene deletions and knock-in were conducted; efficiency was as high as 90% • Through antibiotic resistance repair, ssDNA recombineering parameters were optimized.

Expression of Heterologous OsDHAR Gene Improves Glutathione (GSH)-Dependent Antioxidant System and Maintenance of Cellular Redox Status in Synechococcus elongatus PCC 7942.

An excess of reactive oxygen species (ROS) can cause severe oxidative damage to cellular components in photosynthetic cells. Antioxidant systems, such as the glutathione (GSH) pools, regulate redox status in cells to guard against such damage. Dehydroascorbate reductase (DHAR, EC 1.8.5.1) catalyzes the glutathione-dependent reduction of oxidized ascorbate (dehydroascorbate) and contains a redox active site and glutathione binding-site. The DHAR gene is important in biological and abiotic stress responses involving reduction of the oxidative damage caused by ROS. In this study, transgenic Synechococcus elongatus PCC 7942 (TA) was constructed by cloning the Oryza sativa L. japonica DHAR (OsDHAR) gene controlled by an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible promoter (Ptrc) into the cyanobacterium to study the functional activities of OsDHAR under oxidative stress caused by hydrogen peroxide exposure. OsDHAR expression increased the growth of S. elongatus PCC 7942 under oxidative stress by reducing the levels of hydroperoxides and malondialdehyde (MDA) and mitigating the loss of chlorophyll. DHAR and glutathione S-transferase activity were higher than in the wild-type S. elongatus PCC 7942 (WT). Additionally, overexpression of OsDHAR in S. elongatus PCC 7942 greatly increased the glutathione (GSH)/glutathione disulfide (GSSG) ratio in the presence or absence of hydrogen peroxide. These results strongly suggest that DHAR attenuates deleterious oxidative effects via the glutathione (GSH)-dependent antioxidant system in cyanobacterial cells. The expression of heterologous OsDHAR in S. elongatus PCC 7942 protected cells from oxidative damage through a GSH-dependent antioxidant system via GSH-dependent reactions at the redox active site and GSH binding site residues during oxidative stress.

Tropomyosin: An Excretory/Secretory Protein from Haemonchus contortus Mediates the Immuno-Suppressive Potential of Goat Peripheral Blood Mononuclear Cells In Vitro.

During host-parasite interactions, binding of excretory/secretory proteins (ESPs) on the host immune cells is considered the fundamental phase for regulation of immune responses. In this study, gene encoding Haemonchus contortus tropomyosin (Hc-TpMy), was successfully cloned and expressed, and the recombinant protein after host cell surface attachment was evaluated for immune functional analysis with goat peripheral blood mononuclear cells (PBMCs) in vitro. The isopropyl-β-D-thiogalactopyranoside (IPTG)-induced recombinant protein was successfully recognized by the sera of rat experimentally infected with rHc-TpMy. The immunofluorescence assay detected attachment of rHc-TpMy on the surface of host PBMCs. Furthermore, immunoregulatory roles of rHc-TpMy on cytokines expression, PBMC proliferation, migration, nitric oxide (NO) production, apoptosis and monocytes phagocytosis were observed. The results showed that expression of IL-4 and IFN-γ cytokines, cell proliferation, NO production and PBMC migration were significantly suppressed by goat PBMCs after co-incubation with rHc-TpMy protein. However, the productions of IL-10, IL-17 and TGF-β1 cytokines, PBMCs apoptosis and monocytes phagocytosis were elevated at dose dependent manner. Our findings indicated that rHc-TpMy is an important ES binding protein exhibit distinct immuno-suppressive roles on goat PBMCs which might be a potential molecular target to control haemonchosis in future.

Establishment of toolkit and T7RNA polymerase/promoter system in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a well-known electrogenic bacterium for its respiratory and extracellular electron transfer (EET) capability. However, the genetic toolkits, including promoters, replication origins and biological parts are still rarely used. In this study, constitutive promoters of pLacI, pJ23100, pJ23105, pJ23109, and pTet expressing super-folder green fluorescent protein (sfGFP) were verified in combination with replication origins of pBR322 and p15A. The optimal genetic module was obtained from the pLacI promoter and pBR322 origin, which the specific fluorescence intensity in the MR-1 reached 5518 a.u./g-DCW. T7RNA polymerase (T7RNAP) was also integrated into MR-1 chromosome (i.e., MR1::T7R) by homologous recombination to establish the T7 system. Thus, an expression vector was constructed under T7 promoter and a mobilization gene cluster, which was overexpressed on red fluorescence protein (RFP), carbonic anhydrase (CA), and heme-related proteins. The optimal condition for induction of isopropyl β-d-1-thiogalactopyranoside (IPTG) was determined by the RFP fluorescence intensity, which was 0.5 mM IPTG after 2 h incubation. Moreover, the carbon dioxide capture was enhanced with CAs which activity reached to 12,106 WAU/mg, while the productivity of valuable 5-aminolevuinic acid, a pro-drug for cancer therapy, increased by 3.96-folds with overexpression of HemD in MR1::T7R. The results proved the feasibility of the functional T7 system in Shewanella species.

Transcriptional and Translational Leakiness in Expression of a Fungal Cyclopropane Fatty Acid Synthase in the Escherichia coli pET-vector Expression System

We expressed the putative cyclopropane synthase Cfs1 from the mushroom Coprinopsis cinerea and the native bacterial Cfa enzyme in Escherichia coli as fusion proteins with an Nterminal 10xHis-tag and a proteolytic Factor Xa site, using the commercial pET-16b vector with the T7lac promoter for expression. Proteomics detected both proteins with high Mascot scores via trypsin-digested peptides in total cytoplasmic protein samples from transformed BL21(DE3) bacteria, induced by IPTG (isopropyl β-D-1- thiogalactopyranoside) for T7 RNA polymerase production from theT7 phage gene 1 present in the host genome under control by the lacUV5 promoter. In lower amounts, Cfs1 and Cfa were also found in protein samples from non-induced cells based on the known transcriptional leakiness of the pET-system in expression of T7 RNA polymerase. As a novelty, the results on Cfs1 from proteomics suggest that the sequence at the pET-16b-cfs1 fusion point CTCGAGGAT CCTATG (BamHI cloning site in italic, native cfs1 ATG start codon in bold) serves in transcripts as a minor RBS (ribosome binding site) to inadvertently initiate protein synthesis on the native cfs1 start codon. The first peptide MPAHHHPSSSAPCVSFPSSSK of the native Cfs1 protein was found in E. coli which is only possible if the normal cfs1 ATG was recognized as the start codon in protein biosynthesis. The GAGG-stretch in the above DNA sequence with a 7-bases-aligned spacing to the native cfs1 start codon features a partial Shine-Dalgarno (SD) sequence.

Skimmed milk as an alternative for IPTG in induction of recombinant protein expression

Cost-effectiveness is an important issue in biotechnological manufacturing industry and using alternative cheap materials with the same benefits has been noticed in most literatures. Isopropyl β-d-1-thiogalactopyranoside (IPTG), a well-known chemical element for induction of protein expression, has several disadvantages such as high expense and toxicity. In this study, we aimed to introduce skimmed milk as an alternative material for protein expression by induction of lac operon. In this way, Escherichia coli BL21 (DE3) bacteria were induced using 1 mM IPTG or 1.0% (w/v) skimmed milk. Protein purification was performed using Ni-NTA (nickel-nitrilotriacetic acid) for His-tagged recombinant proteins and protein purity was evaluated by SDS-PAGE. Results showed high level of recombinant protein expression using skimmed milk, and interestingly, the growth rate of bacteria improved. Our findings suggested that skimmed milk can be a suitable alternative for induction of recombinant protein expression, which has advantages such as more availability and affordability, in comparison to IPTG supplementation.

Serodiagnosis of wildlife leptospirosis employing recombinant leptospiral immunoglobulin like b protein antigen

Leptospirosis is a major threat in tropical and subtropical countries as well as temperate countries. The disease is caused by pathogenic Leptospira species and considered to be an emerging or re-emerging disease in many countries of the world. Infection in domestic animals and wildlife can lead to economic loss and pose a potential spread to the communities. In the present study recombinant LigB protein is employed in latex agglutination test, which is a cross reacting lipoprotein able to detect acute infection caused by any pathogenic leptospiral serovars. It was employed for serodiagnosis of leptospirosis. The 46KDa 6X His tagged LigB protein, obtained by IPTG induction of recombinant E. coli M15 cells containing the N-terminal region of LigB gee in P QE30 expression vector, was purified by Ni-NTA affinity chromatography and adsorbed on latex bead surface for performing latex agglutination test against Leptospirosis suspected wildlife field sera. A total of 80 wildlife sera samples were collected, including 27 wild feline sera samples (18 tigers, 8 lions, and 1 jaguar) obtained from Chhatbir zoo, Chandigarh, 42 serasamples ( 8 tigers, 4 lions and 6 leopards, 2 cheethals, 1 black buck, 12 buffaloes and 9 zoo staff) sera and 3 live rodents ) were received from Jodhpur zoo Rajasthan, 8 sera samples (4 tigers, 3 leopards, 1 lion) sera from Van Vihar National park, Bohpal, Madhya Pradesh and 3 sera samples (2 lions,and 1 tiger) received from Biwani Mini zoo, Haryana, India. The result showed that sera were tested positive by rLigB based LAT, which were reconfirmed using microscopic agglutination test (MAT). The results from LAT were in concordance with MAT. In conclusion, rLigB based LAT is a rapid, pen site, reliable diagnostic tool of high sensitivity and specificity, under laboratory and field conditions, for the detection of Leptospirosis .

The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by Escherichia coli Biofilms.

This work assesses the effect of chemical induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) on the expression of enhanced green fluorescent protein (eGFP) by planktonic and biofilm cells of Escherichia coli JM109(DE3) transformed with a plasmid containing a T7 promoter. It was shown that induction negatively affected the growth and viability of planktonic cultures, and eGFP production did not increase. Heterologous protein production was not limited by gene dosage or by transcriptional activity. Results suggest that plasmid maintenance at high copy number imposes a metabolic burden that precludes high level expression of the heterologous protein. In biofilm cells, the inducer avoided the overall decrease in the amount of expressed eGFP, although this was not correlated with the gene dosage. Higher specific production levels were always attained with biofilm cells and it seems that while induction of biofilm cells shifts their metabolism towards the maintenance of heterologous protein concentration, in planktonic cells the cellular resources are directed towards plasmid replication and growth.

Evaluation of soluble expression of recombinant granulocyte macrophage stimulating factor (rGM-CSF) by three different E. coli strains.

Background and purpose:Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine with a wide range of therapeutic applications although expression of GM-CSF in Escherichia coli (E. coli) usually leads to formation of insoluble aggregates mostly lack biological activity. The aim of this study was to compare the soluble expression level of GM-CSF in three E. coli strains, BL21 (DE3), SHuffle® T7 and Origami™ 2 (DE3).Experimental approach:The effect of different temperatures and inducer concentrations on soluble expression of GM-CSF was evaluated. The soluble GM-CSF was subjected to endotoxin removal and purification using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, ultrafiltration. The biological activity of produced GM-CSF was evaluated based on its growth promotion effect on TF-1 cell lines by MTT assay method.Findings / Results:A significant improvement of the soluble yield of GM-CSF (about 30% of GM-CSF was expressed as soluble proteins) was observed when protein expression was induced at 30 °C with 0.5 mM isopropyl β- d-1-thiogalactopyranoside (IPTG) in E. coli Shuffle T7. The soluble GM-CSF with a high purity up to 95 % and specific activity of 1.25 × 104 IU/μg was obtained.Conclusion and implications:The proposed strategy here can be used to improve the soluble expression of other hard-to-express proteins with similar structural properties (i.e., containing disulfide binds or cysteine).

Purification and Optimization of Prokaryotic Expression of CSN5B Protein of <i>Arabidopsis thaliana</i>

CSN5B is a subunit of the COP9 signalosome (CSN) and plays physiological functions in the form of monomer or complex in plants. The CSN5B of Arabidopsis thaliana was cloned by RT-PCR technology and constructed into the prokaryotic expression vector pET-32a (+), which was introduced into E.coli BL21 host bacteria. Isopropylβ-D-1-thiogalactopyranoside (IPTG) was used to induce expression, and the cultural temperature and time was optimized. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was employed to detect the induced recombination protein, and the protein purification was conducted. It was shown that the recombinant CSN5B introduced into E. coli could be induced to express, and the induction was relatively effective in the presence of 1mmol/L of IPTG at 30℃, 4h induction effect is better. The molecular weight of the induced recombinant protein was basically consistent with the theoretical molecular weight. This study provided a theoretical basis for in-depth further exploration of the functions of this gene in the future.

Enhanced 5-Aminolevulinic Acid Production by Co-expression of Codon-Optimized hemA Gene with Chaperone in Genetic Engineered Escherichia coli.

5-Aminolevulinic acid (ALA) is an important metabolic intermediate compound with high value and has recently been used in agriculture and medicine. In this study, we have constructed six recombinant Escherichia coli (E. coli) strains that are involved in pET system under the regulation of the T7 promoter and LacI to express codon-optimized hemA gene from Rhodobacter capsulatus (RchemA) for ALA production via the C4 pathway. Due to codon optimization, hemA has a high transcriptional level; however, most RcHemA proteins were formed as inclusion body. To improve expression in soluble form, the vector with TrxA fusion tag was successfully used and co-expressed with partner GroELS as chaperone in another vector. As a result, ALA production increased significantly from 1.21 to 3.67g/L. In addition, optimal ALA production was developed through adjustment of induction time and isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, as well as substrate addition conditions. By adopting a two-stage induction strategy, the highest ALA reached 5.66g/L when 0.1mM of IPTG was added at early exponential phase (i.e., OD600 was equal to 0.7 to 0.8), while 6g/L of glycine, 2g/L of succinate, and 0.03mM of pyridoxal 5'-phosphate (PLP) were provided in the mid-exponential phase in fermentation.

Early secreted antigenic target of 6-kDa of Mycobacterium tuberculosis induces transition of macrophages into epithelioid macrophages by downregulating iNOS / NO-mediated H3K27 trimethylation in macrophages

BackgroundTuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb). Granuloma is a pathological feature of tuberculosis and is a tight immune cell aggregation caused by Mtb. The main constituent cells are macrophages and their derivative cells including epithelioid macrophages. However, the molecular mechanism of the transition has not been reported. The purpose of this study was to investigate whether early secreted antigenic target of 6-kDa (ESAT6) can induce the transition of bone marrow-derived macrophages (BMDMs) into epithelioid macrophages and its possible molecular mechanism. MethodsThe recombinant ESAT6 protein was obtained from E.coli carrying esat6 gene after isopropyl β-d-thiogalactopyranoside (IPTG) induction. BMDMs were isolated from bone marrow of mice hind legs. Cells viability was detected by Cell Counting Kit 8 (CCK8) assays. The expression levels of mRNA and proteins were detected by qPCR and Western blot, or evaluated by flow cytometry. The expression level of nitric oxide (NO) was measured with a nitric oxide indicator. ResultsESAT6 could significantly induce mRNA and protein expression levels of a group of epithelioid macrophages marker molecules (EMMMs), including E-cadherin, junction plakoglobin, ZO1, desmoplakin, desmoglein3 and catenin porteins, in BMDMs. These events could be abrogated in macrophage from TLR2 deficiency mice. ESAT6 could also markedly induce iNOS/NO production that could significantly inhibit trimethylation of H3K27 in the cells. ESAT6-induced expressions of epithelioid macrophages marker molecules were significantly inhibited in the presence of H3K27 histone demethylase inhibitor GSK J1. Furthermore, ROS scavenging agent N,N'-Dimethylthiourea (DMTU) could markedly inhibit the transition induced by ESAT6 in macrophages. ConclusionThis study demonstrates that ESAT6 bound with TLR2 can activate iNOS/NO and ROS signalings to reduce the trimethylation of H3K27 resulting in the increment of EMMMs expression that is beneficial to the transition of macrophages into epithelioid macrophages. However, hypoxia can inhibit this transition event. This study has provided new evidence of pathogenesis of granuloma caused by Mtb and also proposed new ideas for the treatment of TB.

Highly effective methods for expression/purification of recombinant human HSP90 and its four distinct (N-LR-M-C) domains

Heat shock protein 90 (HSP90) plays essential roles in the normal physiology and comprises four distinct domains, including NH2-terminal (N), charged linker region (LR), middle (M), and COOH-terminal (C) domains, all of which regulate HSP90 biological functions. We reported herein detailed protocols to produce recombinant full-length (FL) and all these four domains of human HSP90 from Escherichia coli. cDNAs encoding FL, N, LR, M and C domains of human HSP90α were amplified and cloned into pET-32b(+) expression vector. All HSP90 constructs were expressed as soluble Trx-His-S tagged proteins after induction with 0.25 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 18 °C overnight and further purified by affinity chromatography using nickel-nitrilotriacetic acid (Ni-NTA) resin. The enterokinase (EK) digestion was optimized for efficient cleavage of the Trx-His-S tag from each HSP90 construct by varying concentrations of EK (0.5–1 U) and urea (0–3 M). Each HSP90 construct was highly purified and approximately 0.1–1 mg proteins were obtained from 100 ml of bacterial culture. All the purified HSP90 constructs were successfully confirmed by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) and their secondary structure was quantified using attenuated total reflection – Fourier-transform infrared (ATR-FTIR) spectroscopy. Our expression and purification protocols would facilitate further structural and functional studies of human HSP90.