OPTIMIZING THE PRODUCTION OF A FUNCTIONAL TYPE A RECOMBINANT ENDOCHITINASE FROM Trichoderma asperellum IN Escherichia coli

Chitinases from the genus Trichoderma fungi are mainly responsible for their anti-fungal activities, which allow them to become the most widely used fungal biocontrol. Therefore, several Trichoderma chitinases have been cloned and expressed to facilitate their production and applications. A previous study of the same authors has characterized an endochitinase from a relatively novel Trichoderma spp., Trichoderma asperellum. To produce this enzyme more economically and efficiently, we reported the synthesis and expression of its synthetic encoding gene in the Escherichia coli M15 strain and established the optimal conditions for preparative scale production of the enzyme in its functional form. By lowering the induction temperatures, we observed substantial improvement in the expression levels of the active enzyme. At 30 oC and 0.5 mM IPTG induction, 1 L of cells yielded approximately 80 - 100 mg of soluble protein, accounting for about 9-11 % of total soluble protein. This figure may be an underestimation of the actual yield, as deduced from the SDS-PAGE data. The recombinant enzyme can be retrieved by simple repeated freezing and thawing cycles and purified to near homogeneity using Ni-NTA chromatography. The purified enzyme showed in vitro colloidal chitin hydrolysis activity. These results could be scaled up to produce soluble 42 kDa chitinase in E. coli. The study demonstrated an economical method to produce chitinases for various agricultural and environmental applications.

Metabolic engineering of Bacillus subtilis with an endopolygalacturonase gene isolated from Pectobacterium. carotovorum; a plant pathogenic bacterial strain

Pectinolytic enzymes or pectinases are synthesized naturally by numerous microbes and plants. These enzymes degrade various kinds of pectin which exist as the major component of the cell wall in plants. A pectinase gene encoding endo-polygalacturonase (endo-PGase) enzyme was isolated from Pectobacterium carotovorum a plant pathogenic strain of bacteria and successfully cloned into a secretion vector pHT43 having σA-dependent promoter for heterologous expression in Bacillus subtilis (WB800N).The desired PCR product was 1209bp which encoded an open reading frame of 402 amino acids. Recombinant proteins showed an estimated molecular weight of 48 kDa confirmed by sodium dodecyl sulphate–polyacrylamide-gel electrophoresis. Transformed B. subtilis competent cells harbouring the engineered pHT43 vector with the foreign endo-PGase gene were cultured in 2X-yeast extract tryptone medium and subsequently screened for enzyme activity at various temperatures and pH ranges. Optimal activity of recombinant endo-PGase was found at 40°C and pH 5.0. To assay the catalytic effect of metal ions, the recombinant enzyme was incubated with 1 mM concentration of various metal ions. Potassium chloride increased the enzyme activity while EDTA, Zn++ and Ca++, strongly inhibited the activity. The chromatographic analysis of enzymatic hydrolysates of polygalacturonic acid (PGA) and pectin substrates using HPLC and TLC revealed tri and tetra-galacturonates as the end products of recombinant endo-PGase hydrolysis. Conclusively, endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis for the first time using pHT43 expression vector and could be assessed for enzyme production using a very simple medium with IPTG induction. These findings proposed that the Bacillus expression system might be safer to escape endotoxins for commercial enzyme production as compared to yeast and fungi. Additionally, the hydrolysis products generated by the recombinant endo-PGase activity offer their useful applications in food and beverage industry for quality products.

Construction of an AI-2 quorum sensing induced heterologous protein expression system in Escherichia coli

Background The pET expression system based on T7 promoter which is induced by isopropyl-β-D-1-thiogalactopyranoside (IPTG) is by far the most commonly used system for production of heterogeneous proteins in Escherichia coli. However, this system was limited by obvious drawbacks including the host toxicity and metabolic burden imposed by the presence of IPTG. Methods In this study, we incorporated the autoinducer-2 (AI-2) quorum sensing system to realize autoinduction of the pET expression system. The autoinduction expression vector pXWZ1 was constructed by inserting the lsr promoter regions into the pET28a(+) vector. The expression efficiency of the reporter genes gfpuv and lacZ by the pXWZ1 and pET28a(+) vectors were compared. Results The results showed that the expression levels of the both report genes in the cells transformed with pXWZ1 without any addition of exogenous inducer were higher than that transformed with pET28a(+) vectors by the induction of IPTG. Conclusion This new auto-induction system will exclude the limitations of the IPTG induction including toxic to host and increasing formation of inclusion body and will become a more economical and convenient tool for recombinant protein expression.

Heat shock transformation and expression of the plasmid containing cytolethal distending toxin of Campylobacter fetus subsp venerealis in Escherichia coli

The cytolethal distending toxins (cdt) is a multi-subunit toxin consisted of three subunit encoded cdtA, cdtB and cdtC. The cdt played an important role as a virulence factor of Campylobacter infection, including C. fetus subsp venerealis. The cdtA which responsible for binding the cdt to cell membrane, was cloned in plasmid expression and inserted into bacterial cells of Escherichia coli BL21(DE3). The research was conducted to evaluate the transformation using the heat shock method of a plasmid containing cdtA3 gene and the protein expression induced by various concentration of IPTG. Transformation was done using the heat shock method at 42oC for 90 second. Evaluation of the transformation was observed on the presence of E. coli BL21(DE3) colonies on Luria Bertani agar containing Ampicillin antibiotic with 100 µg/mL dosage. The recombinant protein was expressed using IPTG-induction with various concentration (0.1mM, 0.25mM, 0.5mM, 0.75mM and 1 mM). The result showed that the transformation and IPTG-induction 0.1 mM produced higher concentration of protein than other concentration applied. The protein characterization was observed with SDS PAGE and cdtA3 protein was detected on 23,4 kDa.

Effects of Lower Temperature on Expression and Biochemical Characteristics of HCV NS3 Antigen Recombinant Protein

The nonstructural antigen protein 3 of the hepatitis C virus (HCV NS3), commonly-used for HCV ELISA diagnosis, possesses protease and helicase activities. To prevent auto-degradation, a truncated NS3 protein was designed by removing the protease domain. Firstly, it was overexpressed in E. coli by IPTG induction under two different temperatures (25 and 37 °C), and purified using affinity chromatography to attain homogeneity above 90%. The molecular mass of purified protein was determined to be approx. 55 kDa. While lowering the temperature from 37 to 25 °C, the yield of the soluble fraction of HCV NS3 was increased from 4.15 to 11.1 mgL−1 culture, which also improved the antigenic activity and specificity. The protein stability was investigated after long-term storage (for 6 months at −20 °C) revealed no loss of activity, specificity, or antigenic efficacy. A thermal stability study on both freshly produced and stored HCV NS3 fractions at both temperatures showed that the unfolding curve profile properly obey the three-state unfolding mechanism. In the first transition phase, the midpoints of the thermal denaturation of fresh NS3 produced at 37 °C and 25 °C, and that produced after long-term storage at 37 °C and 25 °C, were 59.7 °C, 59.1 °C, 55.5 °C, and 57.8 °C, respectively. Microplates coated with the fresh NS3 produced at 25 °C or at 37 °C that were used for the HCV ELISA test and the diagnosis outcome were compared with two commercial kits—Abbott HCV EIA 2.0 and Ortho HCV EIA 3.0. Results indicated that the specificity of the HCV NS3 produced fresh at 25 °C was higher than that of the fresh one at 37 °C, hence showing potential for application in HCV ELISA diagnosis.

A dual prokaryotic (E. coli) expression system (pdMAX)

In this study, we introduced an efficient subcloning and expression system with two inducible prokaryotic expression promoters, arabinose and lac, in a single plasmid in Escherichia coli. The arabinose promoter unit allows for the expression of a FLAG-tagged protein, while the isopropyl-β-D-thiogalactoside (IPTG)-inducible unit allows for the expression of a Myc-tagged protein. An efficient subcloning (DNA insertion) system (iUnit) follows each promoter. The iUnit, based on a toxin that targets DNA topoisomerase of E. coli, allows for effective selection with arabinose or IPTG induction. With the dual promoter plasmid (pdMAX) system, expressed lacZ (β-galactosidase) activity was significantly decreased compared with the original solo expression system. Despite this disadvantage, we believe that the pdMAX system remains useful. A recombinant plasmid (pdMAX/ara/DsRed/IPTG/EGFP; pdMAX/DsRed/EGFP) with DsRed in the arabinose expression unit and EGFP in the IPTG expression unit showed fluorescent protein expression following additional low-temperature incubation. Thus, the novel pdMAX system allowed efficient subcloning of two different genes and can be used to induce and analyze the expression of two distinct genes. The proposed system can be applied to various types of prokaryotic gene expression analysis.

Production of soluble regulatory hydrogenase from Ralstonia eutropha in Escherichia coli using a fed-batch-based autoinduction system

Background Autoinduction systems can regulate protein production in Escherichia coli without the need to monitor cell growth or add inducer at the proper time following culture growth. Compared to classical IPTG induction, autoinduction provides a simple and fast way to obtain high protein yields. In the present study, we report on the optimization process for the enhanced heterologous production of the Ralstonia eutropha regulatory hydrogenase (RH) in E. coli using autoinduction. These autoinduction methods were combined with the EnPresso B fed-batch like growth system, which applies slow in situ enzymatic glucose release from a polymer to control cell growth and protein synthesis rate. Results We were able to produce 125 mg L−1 RH corresponding to a productivity averaged over the whole process time of 3 mg (L h)−1 in shake flasks using classic single-shot IPTG induction. IPTG autoinduction resulted in a comparable volumetric RH yield of 112 mg L−1 and due to the shorter overall process time in a 1.6-fold higher productivity of 5 mg (L h)−1. In contrast, lactose autoinduction increased the volumetric yield more than 2.5-fold and the space time yield fourfold reaching 280 mg L−1 and 11.5 mg (L h)−1, respectively. Furthermore, repeated addition of booster increased RH production to 370 mg L−1, which to our knowledge is the highest RH concentration produced in E. coli to date. Conclusions The findings of this study confirm the general feasibility of the developed fed-batch based autoinduction system and provide an alternative to conventional induction systems for efficient recombinant protein production. We believe that the fed-batch based autoinduction system developed herein will favor the heterologous production of larger quantities of difficult-to-express complex enzymes to enable economical production of these kinds of proteins.

Quantifying the optimal strategy of population control of quorum sensing network in Escherichia coli

Biological functions of bacteria can be regulated by monitoring their own population density induced by the quorum sensing system. However, quantitative insight into the system’s dynamics and regulatory mechanism remain challenging. Here, we construct a comprehensive mathematical model of the synthetic quorum sensing circuit that controls population density in Escherichia coli. Simulations agree well with experimental results obtained under different ribosome-binding site (RBS) efficiencies. We present a quantitative description of the component dynamics and show how the components respond to isopropyl-β-D-1-thiogalactopyranoside (IPTG) induction. The optimal IPTG-induction range for efficiently controlling population density is quantified. The controllable area of population density by acyl-homoserine lactone (AHL) permeability is quantified as well, indicating that high AHL permeability should be treated with a high dose of IPTG, while low AHL permeability should be induced with low dose for efficiently controlling. Unexpectedly, an oscillatory behavior of the growth curve is observed with proper RBS-binding strengths and the oscillation is greatly restricted by the bacterial death induced by toxic metabolic by-products. Moreover, we identify that the mechanism underlying the emergence of oscillation is determined by the negative feedback loop structure within the signaling. Bifurcation analysis and landscape theory are further employed to study the stochastic dynamic and global stability of the system, revealing two faces of toxic metabolic by-products in controlling oscillatory behavior. Overall, our study presents a quantitative basis for understanding and new insights into the control mechanism of quorum sensing system, providing possible clues to guide the development of more rational control strategy.

Metabolic engineering of Bacillus subtilis with an Endopolygalacturonase gene Isolated from Pectobacterium carotovorum; a Plant Pathogenic bacterial strain.

Pectinolytic enzymes [pectinases] produced by microbes are highly important for their biotechnological use in processing of vegetables and fruits beverages and use in pulp and paper industry. A pectinases, namely endo-polygalacturonase [endo-PGase], encoding gene isolated from Pectobacterium carotovorum, a plant pathogenic strain of bacteria was successfully cloned into a secretion vector pHT43 having σ?-dependent promoter P grac . For enhanced expression analysis, competent cells of Bacillus subtilis (WB800N) were prepared at stationary phase using high salt medium. The recombinant B. subtilis competent cells, harboring the engineered pHT43 with the endo-PGase gene were cultured in 2X-yeast extract tryptone medium. The recombinant endo-PGase enzyme was secreted directly into the medium after 72 hours of the first IPTG induction. The recombinant endo-PGase was screened for its activity at various temperatures and pH ranges. Optimal activity was found at pH 5.0 and a temperature of 40°C with a stability ranging from pH 5.0-9.0. For detection of metal ion effect, recombinant enzyme was incubated with 1mM concentration of; Ca ++ , Mg ++ , Zn ++ , EDTA, K ++ for 45 minutes. Resultantly, Ca ++ , EDTA and Zn ++ strongly inhibited the enzyme activity. The chromatographic analysis of enzymatic hydrolysate of polygalacturonic acid [PGA] and pectin substrates using HPLC and TLC revealed that tri and tetra-galacturonates were the end products of hydrolysis. The study led to the conclusion that endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis and assessed for enzyme production using a very simple medium with IPTG induction. These findings proposed that the Bacillus expression system might be safe for commercial enzyme production as compared to yeast and fungi to escape endotoxins.

Optimization of Monochloroacetic Acid Biodegradation by Recombinant E. coli BL21 (DE3)/pET-bcfd1 Carrying Haloacid Dehalogenase Gene from Bacillus cereus IndB1

In recent years, attention to microbial dehalogenase has continually increased due to its potential application, both in bioremediation and in the biosynthesis of fine chemicals. Many microbial recombinant strains carrying dehalogenase gene have been developed, particularly to increase the dehalogenase production and its quality. In this study, we aimed to find the optimum condition for the production of active haloacid dehalogenase by E. coli BL21 (DE3) harboring recombinant plasmid pET-bcfd1 that carried haloacid dehalogenase gene from Bacillus cereus IndB1 local strain. This would be examined by assessing the ability of whole cell life culture to degrade monochloroacetic acid (MCA) and quantifying the chloride ion released into the medium. Several variables were evaluated to find this optimal condition. We found that the best condition for MCA biodegradation using this recombinant clone was at 0.2 mM MCA, 10 μM of isopropyl β-D-1-thiogalactopyranoside (IPTG), 6 hours of pre-induction incubation at 37ºC with shaking, 2 hours IPTG induction at 30ºC with shaking, at pH 7 in Luria Bertani (LB) liquid medium without NaCl, which produced about 0.056 mM chloride ions. Inducer concentration, pre-induction incubation time and temperature, as well as induction time and temperature were apparent to be associated with the expression of the protein, while the MCA concentration and the pH of the medium influenced the ability of the recombinant E. coli BL21 (DE3)/pET-bcfd1 to grow in toxic environment. Our findings laid the foundation for exploration of dehalogenases from local Bacillus strains through genetic engineering for MCA biodegradation