IPTG Concentration Research Articles

Molecular Characterization, Structural Modeling, and Evaluation of Antimicrobial Activity of Basrai Thaumatin-Like Protein against Fungal Infection.

A thaumatin-like protein gene from Basrai banana was cloned and expressed in Escherichia coli. Amplified gene product was cloned into pTZ57R/T vector and subcloned into expression vector pET22b(+) and resulting pET22b-basrai TLP construct was introduced into E. coli BL21. Maximum protein expression was obtained at 0.7 mM IPTG concentration after 6 hours at 37°C. Western blot analysis showed the presence of approximately 20 kDa protein in induced cells. Basrai antifungal TLP was tried as pharmacological agent against fungal disease. Independently Basrai antifungal protein and amphotericin B exhibited their antifungal activity against A. fumigatus; however combined effect of both agents maximized activity against the pathogen. Docking studies were performed to evaluate the antimicrobial potential of TLP against A. fumigatus by probing binding pattern of antifungal protein with plasma membrane ergosterol of targeted fungal strain. Ice crystallization primarily damages frozen food items; however addition of antifreeze proteins limits the growth of ice crystal in frozen foods. The potential of Basrai TLP protein, as an antifreezing agent, in controlling the ice crystal formation in frozen yogurt was also studied. The scope of this study ranges from cost effective production of pharmaceutics to antifreezing and food preserving agent as well as other real life applications.

Soluble expression, purification and structural analysis of the bHLH transcription factor Bmsage of Bombyx mori

Basic helix loop helix (bHLH) transcription factor plays an important role in biological processes. Bmsage is a class of bHLH transcription factor highly expressed in the silk gland of Bombyx mori, which is not only involved in the developmental regulation of the silk gland cells at the embryonic period, but also plays a crucial regulatory role during the synthesis of silk protein. However, currently, much of the property and structure of Bmsage is still remained unknown. To study the property, structure and biological role of Bmsage, we constructed several prokaryotic expression vectors of Bmsage fused with NusA, MBP, SUMO, Trx and His tags, respectively, then screened and determined the best soluble expression vector and condition of Bmsage in Escherichia coli combining with the induction temperature and IPTG concentration, and further purified the recombinant Bmsage by Ni-column affinity chromatography according to the established expression condition and characterized its secondary structure using circular dichroism spectra. The results showed that NusA and MBP could significantly enhance the soluble expression of Bmsage in E. coli, but it was difficult to separate Bmsage from these tags. SUMO could not only increase the soluble expression of Bmsage in E. coli to a certain degree, but also be effectively separated from Bmsage. Other tags did not effectively promote the soluble expression of Bmsage in E. coli. Circular dichroism spectra showed that the purified Bmsage had well-defined α-helix structure in solution, indicating that SUMO may promote the correct folding of Bmsage into native-like structure. These work not only establish a foundation for further study of the property, structure and function of Bmsage, but also provide a reference for the expression and purification of other similar proteins.

Expression and purification of recombinant TAT-BoNT/A(1–448) under denaturing and native conditions

ABSTRACTBotulinum toxin type A can temporarily inhibit muscle contraction. Currently, physicians administer this toxin as a bio-drug in treatment of some muscle contraction disorders. TAT-BoNT/A(1–448) is a functional recombinant protein derived from botulinum toxin light chain. Unlike the full length botulinum toxin, TAT-BoNT/A(1–448) is a self-permeable molecule which can pass through bio-surfaces so can be used as a topical therapeutic agent without injection. To maintain the functionality of TAT-BoNT/A(1–448), it is necessary to restore its normal folding upon expression and purification. In this study, we have investigated and optimized expression conditions for this novel recombinant protein. Under denaturing condition (1 mM IPTG, at 37°C), the chimeric protein was produced as inclusion body and required to be purified using denaturing agents (e.g. urea). Yet, lower incubation temperature (18°C) and less IPTG concentration (0.5 mM) induce a protein under native condition. In such condition, about 60% of the chimeric protein was expressed in soluble form.

Bovine Sex Determining Region Y: Cloning, Optimized Expression, and Purification

ABSTRACTSex determining region Y gene (SRY) is located on Y chromosome and encodes a protein with 229 amino acids. In this study, ORF region of SRY with a length of 690 bp was synthesized using PCR and ligated to pET28a (+), then transformed in E.coli DH5α. E.coli BL21 (DE3) strain was chosen to express recombinant bovine SRY protein. A set of optimization steps was taken including different concentrations of IPTG, glucose, and temperatures at differed incubation times after the induction. Results showed that temperature points and different concentrations of IPTG and glucose had a significant effect (p < 0.01) on total protein and recombinant bovine SRY. After purification, various temperatures and concentrations of IPTG showed meaningful effects (p < 0.01) on the solubility of expressed recombinant SRY. Highest soluble rSRY protein amount was achieved where 0.5 mM IPTG and 0.5% glucose was used at 20°C during induction. In the absence of glucose, the highest amount of soluble recombinant SRY levels were achieved at the concentrations of 0.8 mM of IPTG at 28°C, 20°C, and 1.5 mM IPTG at 37°C during induction for 16, 24, and 8 hours, respectively. Regarding the results obtained in this study, it could be stated that by decreasing temperature and inducer concentration, soluble bovine SRY protein expression increases.

Expression and Secretion of Endostar Protein by Escherichia Coli: Optimization of Culture Conditions Using the Response Surface Methodology

Endostar as a specific drug in treatment of the nonsmall cell lung cancer is produced using Escherichia coli expression system. Plackett-Burman design (PBD) and response surface methodology (RSM) are statistical tools for experimental design and optimization of biotechnological processes. This investigation aimed to predict and develop the optimal culture condition and its components for expression and secretion of endostar into the culture medium of E. coli. The synthetic endostar coding sequence was fused with PhoA signal peptide. The nine factors involved in the production of recombinant protein-postinduction temperature, cell density, rotation speed, postinduction time, concentration of glycerol, IPTG, peptone, glycine, and triton X-100-were evaluated using PBD. Four significant factors were selected based on PBD results for optimizing culture condition using RSM. Endostar was purified using cation exchange chromatography and size exclusion chromatography. The maximum level of endostar was obtained under the following condition: 13.57-h postinduction time, 0.76% glycine, 0.7% triton X-100, and 4.87% glycerol. The predicted levels of endostar was significantly correlated with experimental levels (R 2=0.982, P=0.00). The obtained results indicated that PBD and RSM are effective tools for optimization of culture condition and its components for endostar production in E. coli. The most important factors in the enhancement of the protein production are glycerol, glycine, and postinduction time.

High level extracellular production of a truncated alkaline β-mannanase from alkaliphilic Bacillus sp. N16-5 in Escherichia coli by the optimization of induction condition and fed-batch fermentation.

To improve the extracellular production of alkaline β-mannanase from alkaliphilic Bacillus sp. N16-5 in Escherichia coli, two truncated recombinant mannanases (32a-ManAR2 and 22b-ManAR2) were obtained. Compared with the full-length mannanases (32a-ManAR1 and 22b-ManAR1), the truncated mannanases not only showed higher secretion rate, but also exhibited higher thermostability and alkalistability. The K m value (11mg/mL) of 32a-ManAR2 was higher than that (1.46mg/mL) of 32a-ManAR1. The specific activity of 22b-ManAR2 was 2.7 times higher than that of 22b-ManAR1. 22b-ManAR2 showed the highest k cat/K m value of 602.7ml/mgs. The parameters of induction for recombinant mannanase production of E. coli BL21 (pET32a-manAR2) and E. coli BL21 (pET22b-manAR2) were subsequently optimized. The yield of soluble mannanase was found to be enhanced with lower induction temperature (25°C), lower IPTG concentration (0.01-0.05mM), and Triton X-100 supplement (0.1%) in a shake flask. Moreover, a one-time feeding strategy and Triton X-100 supplement were applied in production of 22b-ManAR2 in a 10L fermentor. The productivity of the total soluble mannanase reached 9284.64U/mL with the extracellular rate of 74% at 46h of fermentation, which was the highest productive level of alkaline β-mannanase in recombinant E. coli to date.

Cloning, expression, and purification of recombinant major mango allergen Man i 1 in Escherichia coli

In recent years, the number of people around the world who suffer from fruit allergies has increased. Mango can induce anaphylaxis, and two major mango allergens have been identified – Man i 1 and Man i 2. Apart from their molecular weights and pI values, no other information about them is known. This work identifies the DNA and amino acid sequences of Man i 1 and constructs an expression system for recombinant Man i 1 (rMan i 1). Firstly, 3′ and 5′ RACE assays were used to identify the cDNA fragment of Man i 1. Subsequently, the full length of Man i 1 cDNA was inserted into a pET-21a(+) vector, and the inserted plasmid was transformed to Escherichia coli BL21 (DE3) to express rMan i 1. The conditions for the expression of rMan i 1, including IPTG concentration, induction temperature, and induction time, were optimized. The highest amount of soluble rMan i 1 was obtained after induction with 0.1 mM IPTG at 16 °C for 20 h. The His-tagged rMan i 1 was purified using Ni-NTA agarose and its identity was verified using an anti-histidine antibody and the serum of a mango-allergic person. Additionally, rMan i 1 was identified as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and shared 86.2% identity in amino acid sequence of GAPDH from wheat. Finally, an E. coli expression system of rMan i 1 was established, with the potential to be used in immunotherapy against mango allergy or the development of assays for detecting the residues of mango allergens.

Fed-batch production of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in soluble form in Escherichia coli and its purification and characterization

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent. The aim of this study is to produce large quantities of highly pure and bioactive recombinant human TRAIL. Here, TRAIL was expressed in soluble form by pH-stat fed-batch cultivation and purified using a rapid and simple two-step chromatographic procedure. To improve the soluble yield, expression of TRAIL in Escherichia coli was induced with low IPTG concentration (0.1 mM) at low temperature (28 °C) supplemented with ZnSO4 (0.5 mM), using glycerol as carbon source. Under the optimized conditions, 4.14 ± 0.19 g/L of TRAIL in soluble form was achieved at 19 h without pure oxygen. To purify the recombinant TRAIL, we developed an efficient two-step chromatographic procedure including affinity chromatography and cation-exchange chromatography, especially improved the cation-exchange chromatography using a combination of pH and NaCl gradients strategy. Consequently, 4313.5 mg of target protein with high purity (98.1%) was obtained from 2.3 L of cell broth. Our results also showed that the purified TRAIL was with ordered secondary and tertiary structures, in homogeneous form and with strong cytotoxicity.

Light-induced gene expression with photocaged IPTG for induction profiling in a high-throughput screening system.

BackgroundInducible expression systems are frequently used for the production of heterologous proteins. Achieving maximum product concentrations requires induction profiling, namely the optimization of induction time and inducer concentration. However, the respective experiments can be very laborious and time-consuming. In this work, a new approach for induction profiling is presented where induction in a microtiter plate based cultivation system (BioLector) is achieved by light using photocaged isopropyl β-d-1-thiogalactopyranoside (cIPTG).ResultsA flavin mononucleotide-based fluorescent reporter protein (FbFP) was expressed using a T7-RNA-polymerase dependent E. coli expression system which required IPTG as inducer. High power UV-A irradiation was directed into a microtiter plate by light-emitting diodes placed above each well of a 48-well plate. Upon UV irradiation, IPTG is released (uncaged) and induces product formation. IPTG uncaging, formation of the fluorescent reporter protein and biomass growth were monitored simultaneously in up to four 48-well microtiter plates in parallel with an in-house constructed BioLector screening system. The amount of released IPTG can be gradually and individually controlled for each well by duration of UV-A exposure, irradiance and concentration of photocaged IPTG added at the start of the cultivation. A comparison of experiments with either optical or conventional IPTG induction shows that product formation and growth are equivalent. Detailed induction profiles revealed that for the strain and conditions used maximum product formation is reached for very early induction times and with just 6–8 s of UV-A irradiation or 60–80 µM IPTG.ConclusionsOptical induction and online monitoring were successfully combined in a high-throughput screening system and the effect of optical induction with photocaged IPTG was shown to be equivalent to conventional induction with IPTG. In contrast to conventional induction, optical induction is less costly to parallelize, easy to automate, non-invasive and without risk of contamination. Therefore, light-induced gene expression with photocaged IPTG is a highly advantageous method for the efficient optimization of heterologous protein production and has the potential to replace conventional induction with IPTG.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0461-3) contains supplementary material, which is available to authorized users.

Optimization of cold-adapted alpha-galactosidase expression in Escherichia coli

α-Galactosidase (α-PsGal) of the cold-adapted marine bacterium Pseudoalteromonas sp. KMM 701 was cloned into the pET-40b(+) vector to study its properties and to develop an effective method for modifying human B-erythrocytes into O-blood group. The use of heat-shock as a pre-induction treatment, IPTG concentration of 0.2 mM and post-induction cultivation at 18 °C for 20 h in the developed MX-medium allowed increasing the recombinant Escherichia coli Rosetta (DE3)/40Gal strain productivity up to 30 times and the total soluble α-PsGal yield up to 40 times.

Evaluation of the Expression of Recombinant Type A Botulinum Neurotoxin Light Chain Loaded onto a Cell-Penetrating Peptide

BACKGROUND AND OBJECTIVE: Botulinum toxin type A (BoNT/A) is widely used in the treatment of some muscle contraction disorders through injection. This study aimed to produce recombinant protein through covalent bonding of the light chain of BoNT/A to the peptide TAT (47-57), and to create an optimized condition for expression and purification of the protein. METHODS: In this preliminary study, the nucleotide sequences of the light chain of BoNT/A and TAT peptide were obtained from Gen Bank, and genetic structures containing these sequences were designed and engineered. After cloning into the BL21 (DE3) E. coli vector, expression was induced by IPTG. Thereafter, optimum thermal condition and IPTG concentration for maximum expression were determined based on the difference in the intensity of staining between the bands on SDS-PAGE protein gel. The recombinant protein was purified through nickel column chromatography (Ni-NTA). FINDINGS: The produced chimeric protein is insoluble in normal conditions (1 mM IPTG and at 37˚ C). Through optimization of expression conditions (0.5 mM IPTG and at 18˚ C) 60% of the chimeric protein was produced as solution. CONCLUSION: Based on our results, through expressing the recombinant protein as a solution, the protein maintained its proper folding and function. Hence, the use of denaturation compounds for solution making and destabilization of folded protein structures is not required.

Glucosylation of aroma chemicals and hydroxy fatty acids

To explore the utility of glycosyltransferases as novel biocatalysts, we isolated the glycosyltransferase genes CaUGT2 and SbUGTA1 from Catharanthus roseus and Starmerella bombicola, respectively and heterologously expressed them in Escherichia coli. The purified recombinant proteins were assayed with a variety of small molecule substrates. Carvacrol and its phenol isomer thymol are efficiently glucosylated by CaUGT2. The Vmax/Km ratios show that CaUGT2 exhibits the highest specificity towards carvacrol, followed by thymol, geraniol, eugenol, vanillin, menthol, and tyrosol. In contrast, SbUGTA1 accepts ω-hydroxy fatty acids and 1-alkanols as substrates. The Vmax/Km ratios indicate that SbUGTA1 exhibits the highest specificity towards 16-hydroxy palmitic acid, followed by octanol, decanol, and hexadecanol. In biotransformation experiments 23, 88 and 99% of octanol, 16-hydroxy palmitic acid, and decanol, respectively is converted into the corresponding β-glucosides by E. coli cells expressing SbUGTA1 whereas those cells expressing CaUGT2 glucosylate 18, 61, 77 and 97% of applied eugenol, thymol, vanillin, and carvacrol, respectively. To optimize the biotransformation rate, the effects of the concentration of IPTG, glucose, and substrate on the production of glucosides were tested. Taken together, this procedure is a simple operation, environmentally friendly, and is useful for the preparation of glycosides as additives for food and cosmetics.

A Liquid-Based Colorimetric Assay of Lysine Decarboxylase and Its Application to Enzymatic Assay.

A liquid-based colorimetric assay using a pH indicator was introduced for high-throughput monitoring of lysine decarboxylase activity. The assay is based on the color change of bromocresol purple, measured at 595 nm in liquid reaction mixture, due to an increase of pH by the production of cadaverine. Bromocresol purple was selected as the indicator because it has higher sensitivity than bromothymol blue and pheonol red within a broad range and shows good linearity within the applied pH. We applied this for simple determination of lysine decarboxylase reusability using 96-well plates, and optimization of conditions for enzyme overexpression with different concentrations of IPTG on lysine decarboxylase. This assay is expected to be applied for monitoring and quantifying the liquid-based enzyme reaction in biotransformation of decarboxylase in a high-throughput way.

Expression of hCA IX isoenzyme by using sumo fusion partner and examining the effects of antitumor drugs / Sumo füzyon partneri kullanarak hCA IX izoenziminin ekspresyonu ve antitümör ilaçların etkilerinin incelenmesi

Abstract Objective: In this study, investigating the effects of inhibition of the enzyme activity of some antitumor drugs and the Cancer-Related Human Carbonic Anhydrase IX (hCA IX) isoenzyme expressing as a SUMO fusion protein in an Escherichia coli expression system were aimed. Methods: hCA IX isoenzyme was expressed using SUMO fusion technology. The fusion protein was expressed in a totally soluble form and the expression was verified by SDS-PAGE analysis. Affinity chromatography was used in the purification processes. The effects of certain antitumor drugs on enzyme activity were investigated in vitro conditions by using esterase activity. IC50 values of drugs showing the inhibitory effect were calculated. Inhibition types and Ki values for antitumor drugs, which inhibit the enzyme, were determined by separately plotting Lineweaver- Burk plots. Results: The molecular weight of the fusion protein was approximately 85kDa. The optimal induction concentration of IPTG and the growth temperature were found to be 1.0mM and 30°C. The fusion protein was purified at approximately 3.07-fold with a yield of 92.58%, and a specific activity of 43707EU/mg proteins by nickel nitrilo-triacetic acid resin chromatography. Conclusion: Our work is extremely important because CA IX plays a clinical role as a biomarker in cancer diagnosis and the use of specific inhibitors of the CA IX enzyme will be useful in the fight against cancer. In vitro inhibition studies on the recombinant hCA IX enzyme can shed light on the development of anticancer drugs for cancers overexpressing CA IX.

Exploring the role of sigma factor gene expression on production by Corynebacterium glutamicum: sigma factor H and FMN as example.

Bacteria are known to cope with environmental changes by using alternative sigma factors binding to RNA polymerase core enzyme. Sigma factor is one of the targets to modify transcription regulation in bacteria and to influence production capacities. In this study, the effect of overexpressing each annotated sigma factor gene in Corynebacterium glutamicum WT was assayed using an IPTG inducible plasmid system and different IPTG concentrations. It was revealed that growth was severely decreased when sigD or sigH were overexpressed with IPTG concentrations higher than 50 μM. Overexpression of sigH led to an obvious phenotypic change, a yellow-colored supernatant. High performance liquid chromatography analysis revealed that riboflavin was excreted to the medium when sigH was overexpressed and DNA microarray analysis confirmed increased expression of riboflavin biosynthesis genes. In addition, genes for enzymes related to the pentose phosphate pathway and for enzymes dependent on flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), or NADPH as cofactor were upregulated when sigH was overexpressed. To test if sigH overexpression can be exploited for production of riboflavin-derived FMN or FAD, the endogenous gene for bifunctional riboflavin kinase/FMN adenyltransferase was co-expressed with sigH from a plasmid. Balanced expression of sigH and ribF improved accumulation of riboflavin (19.8 ± 0.3 μM) and allowed for its conversion to FMN (33.1 ± 1.8 μM) in the supernatant. While a proof-of-concept was reached, conversion was not complete and titers were not high. This study revealed that inducible and gradable overexpression of sigma factor genes is an interesting approach to switch gene expression profiles and to discover untapped potential of bacteria for chemical production.

Development of Pgrac100-based expression vectors allowing high protein production levels in Bacillus subtilis and relatively low basal expression in Escherichia coli.

BackgroundIn general, fusion of recombinant genes to strong inducible promoters allowing intracellular expression in Bacillus subtilis is a two-step process. The ligation products are transformed into Escherichia coli, followed by identification of the correct plasmid, and this plasmid is subsequently transformed into B. subtilis. This raises the problem that basal level of expression of the recombinant gene could be harmful for E. coli cells. Based on the Pgrac promoter, we optimized the UP element, the -35, 15, -10 and the +1 region to enhance the promoter activity in B. subtilis after induction. However, detailed investigations for a promoter to develop expression vectors that allows high protein production levels in B. subtilis and a relatively low basal expression levels in E. coli has not been studied yet.ResultsWe screened the previously constructed library of E. coli – B. subtilis shuttle vectors for high level expression in B. subtilis and low basal level in E. coli. Promoter Pgrac100 turned out to meet these criteria, in which ß-galactosidase expression level of Pgrac100-bgaB is about 9.2 times higher than Pgrac01-bgaB in B. subtilis and the ratio of those in induced B. subtilis over un-induced E. coli from Pgrac100-bgaB is 1.3 times higher than Pgrac01-bgaB. Similarly, GFP expression level of Pgrac100-gfp is about 27 times higher than that of Pgrac01-gfp and the ratio from Pgrac100-gfp is 35.5 times higher than Pgrac01-gfp. This promoter was used as a basis for the construction of three novel vectors, pHT253 (His-tag-MCS), pHT254 (MCS-His-tag) and pHT255 (MCS-Strep-tag). Expression of the reporter proteins BgaB and GFP using these expression vectors in B. subtilis at a low IPTG concentration were measured and the fusion proteins could be purified easily in a single step by using Strep-Tactin or IMAC-Ni columns.ConclusionsThis paper describes the construction and analysis of an IPTG-inducible expression vector termed Pgrac100 for the high level production of intracellular recombinant proteins in B. subtilis and a relatively low basal expression level in E. coli. Based on this vector, the derivative vectors, Pgrac100-His-tag-MCS, Pgrac100-MCS-His-tag and Pgrac100-MCS-Strep-tag have been constructed.

Optimization of expression and purification of HSPA6 protein from Camelus dromedarius in E. coli

The HSPA6, one of the members of large family of HSP70, is significantly up-regulated and has been targeted as a biomarker of cellular stress in several studies. Herein, conditions were optimized to increase the yield of recombinant camel HSPA6 protein in its native state, primarily focusing on the optimization of upstream processing parameters that lead to an increase in the specific as well as volumetric yield of the protein. The results showed that the production of cHSPA6 was increased proportionally with increased incubation temperature up to 37°C. Induction with 10μM IPTG was sufficient to induce the expression of cHSPA6 which was 100 times less than normally used IPTG concentration. Furthermore, the results indicate that induction during early to late exponential phase produced relatively high levels of cHSPA6 in soluble form. In addition, 5h of post-induction incubation was found to be optimal to produce folded cHSPA6 with higher specific and volumetric yield. Subsequently, highly pure and homogenous cHSPA6 preparation was obtained using metal affinity and size exclusion chromatography. Taken together, the results showed successful production of electrophoretically pure recombinant HSPA6 protein from Camelus dromedarius in Escherichia coli in milligram quantities from shake flask liquid culture.

Over-expression and Purification of Recombinant 3C-like Protease from Black Queen Cell Virus in Escherichia coli

Black queen cell virus is one of the members belonging to Picornavirus, which possesses a 3C-like protease. Viral replication and capsid assembly in the order Picornavirales require polyprotein proteolysis processing, which is performed by 3C or 3C-like (3CL) proteases. Therefore, viral proteases are attractive targets for anti-viral therapy. The complete 3C-like protease gene from Black queen cell virus was cloned into pGEM-3Zf(+) vector, and the amino acid sequence revealed a high homology (99.1%) to 3C-like protease deposited in a reference Genbank EF517515.1. For the expression, 3C-like protease gene was subcloned into pMAL-C2 vector system. The expression level of MBP-BQCV-3CL protein was evaluated under different standards of cell optical density, IPTG concentration, induction time, and induction temperature. As the result, the optimal condition was confirmed as the induction with 0.1mM IPTG at 25°C for 6 hours, and a purification step using amylose resin reached a high protein yield of approximately 2.22mg per 1ml of cultured cells. Purified MBP-BQCV-3CL proteins can provide an important antigen source to generate monoclonal antibody and will become a potential candidate for anti-viral drugs, which can inactivate 3C-like protease function as well as BQCV replication.

Cloning and prokaryotic expression analysis of squalene synthase 2 (SQS2) from Salvia miltiorrhiza f. alba

According to the designed specific primers of gene fragment based on the Salvia miltiorrhiza transcriptome data, a full-length cDNA sequence of SQS2 from S. miltiorrhiza f. alba was cloned by the method of reverse transcription polymerase chain reaction (RT-PCR). The SmSQS2 cDNA sequence was obtained, this sequence is named SmSQS2 and its GenBank registration number is KM244731. The full length of SmSQS2 cDNA was 1245 bp, encoding 414 amino acids including 5'UTR 115 bp and 3'UTR 237 bp. Sequence alignment and phylogenetic analysis demonstrated that SmSQS2 had relative close relationship to the SQS2 of S. miltiorrhiza. The induction of E. coli [pET28-SQS2] in different temperature, induction time, IPTG concentrations and density of inducing host bacterium (A600) were performed, Shaking the culture at 30 degrees C until the A600 is approximately 0.6 and add IPTG to final concentration of 0.2 mmol x L(-1), and then the optimal expression of SmSQS2 recombinant protein were accumulated after the induction time of 20 h. The research provided important base for the study of sterol and terpene biosynthesis of SQS2 in S. miltiorrhiza f. alba.

An ApnAase / mRNA Decapping Nudix Hydrolase from Mycobacterium leprae

The diadenosine polyphosphatases (ApnAases) / mRNA decapping enzymes are a family of enzymes within the Nudix hydrolase superfamily. The diadenosine polyphosphatases from Legionella pneumophilia and Bartonella bacilliformis have been found to be important in each pathogen's ability to invade its host cells. An ApnAases / mRNA decapping enzyme from Mycobacterium tuberculosis has been characterized in our laboratory and a homolog to this enzyme has been uncovered in Mycobacterium leprae. We have cloned and expressed the M. leprae homolog and have determined that it is an ApnAase. While the enzyme has good expression, it is rather insoluble. We have lowered the expression temperature, lowered the concentration of IPTG, incorporated GroESL, and used a Rosetta cell line to try to increase solubility, but without much effect. We are still working to increase solubility, before we purify and further characterize the enzyme. If these Mycobacteria ApnAases / mRNA decapping enzymes are found to be involved in invasiveness and thus in virulence, then these enzymes could be potential novel antibiotic targets in M. tuberculosis and M. leprae. This research was supported by a NIH AREA Grant and a RIT Graduate Student Grant.