Coli Host Research Articles

Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach.

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial populations. This is especially the case under rapidly changing environments such as fluctuating antibiotics exposure. We recently showed that plasmids maintain antibiotic resistance genes in Escherichia coli without positive selection for the plasmid presence. Here we describe an experimental system that allows following both the plasmid genotype and phenotype in long-term evolution experiments. We use molecular techniques to design a model plasmid that is subsequently introduced to an experimental evolution batch system approach in an E. coli host. We follow the plasmid frequency over time by applying replica plating of the E. coli populations while quantifying the antibiotic resistance persistence. In addition, we monitor the conformation of plasmids in host cells by analyzing the extent of plasmid multimer formation by plasmid nicking and agarose gel electrophoresis. Such an approach allows us to visualize not only the genome size of evolving plasmids but also their topological conformation-a factor highly important for plasmid inheritance. Our system combines molecular strategies with traditional microbiology approaches and provides a set-up to follow plasmids in bacterial populations over a long time. The presented approach can be applied to study a wide range of mobile genetic elements in the future.

Broad-host-range application of the srfA promoter from Bacillus subtilis in Escherichia coli

The promoter of the srf operon (PsrfA) had been used to construct a cell-density-dependent expression system in B. subtilis in our previous work. The PsrfA and its derivative PsrfA12 showed good performance of heterologous protein expression in B. subtilis. In this work, using green fluorescent protein (GFP) and β-galactosidase (LacZ) as the reporter proteins, the host feasibility and expression characteristics of the PsrfA and PsrfA12 in E. coli were identified. The prominent green fluorescence shooted by laser scanning confocal microscope, fluorescence intensity measured by spectrophotometer and the distinct protein bands detected by SDS-PAGE demonstrated that the GFP could be largely expressed under the control of the PsrfA and PsrfA12 in the E. coli host strain of BL21 (DE3) and JM109 and the expression of GFP in strain BL21 (DE3) was much higher than that of in strain JM109. Meanwhile, the promoter PsrfA 12 was much stronger than PsrfA to the extent that the GFP controlled by PsrfA12 in strain BL21 (DE3) was leaked into the supernatant. And the fluorescence intensity detected in the supernatant of the recombinant strain BL21 (DE3) containing PsrfA12 was 10.25-fold higher than that of strain JM109 containing PsrfA. Moreover, the LacZ could also be produced by PsrfA and PsrfA12 in strain BL21 (DE3) and JM109 and the strain JM109 showed better performance than BL21 (DE3) in expressing LacZ. The LacZ activity controlled by PsrfA and PsrfA12 in JM109 were separately 2.47-fold and 2.36-fold higher than that of in strain BL21 (DE3). This work will broaden the applied range of the PsrfA and enrich the efficient toolbar for cross-species gene expression or module construction in E. coli and B. subtilis.

Construction and Optimization of Prethrombin-2 Human Genes in E. coli for the Production of Active Thrombin

Prothrombin-2 is a thrombin precursor, which plays an essential role in the conversion of fibrinogen to fibrin during the blood clotting process. In this study, we designed and constructed the human prethrombin-2 (hPT2) gene. hPT2 gene fused with a tag at the n terminal position, which contains the sequence intein followed by chitin binding domain (CBD) that useful for the purification process. The digestion process of hPT2 tag induced by changing its pH/temperature. The hPT2 codon that used in this study designed according to the preference codon of E. coli. hPT2 gene was created using the OPTIMIZER software with the addition of two side restriction at the 5’ and 3’, BamHI and XhoI respectively then were cloned by using the pMAT vector in E. coli. Moreover, hPT2 fragment ligated into an expression vector, pTWIN1, for E. coli. The result from the characterization hPT2 gene in pTWIN1 that using DNA sequencing method shown that the designed hPT2 gene cloned. Thus, the cloned hPT2 gene used as a precursor for PT2 expression in the E. coli host.

Structural and functional evaluation of recombinant histidine phosphokinase NisK and response regulator NisR: in silico and experimental approach

In the two-component system of NisRK from Lactococcus lactis, the production of nisin is affected by transmembrane NisK and activation of intracellular NisR. The transcription of nisin structural genes can be induced by derivatives of nisin. NisR activation leads to the activation of nisA/Z transcription, which encodes the nisin maturation machinery, nisin regulation and activation of the nisFEG operon to confer immunity. The aim of this study was to express the Lactococcus lactis histidine phosphokinase NisK and response regulator NisR in E. coli, and to perform activity assays and in silico analysis. In silico methods were applied to study the properties and structures of the NisK and NisR proteins, including prediction of physicochemical characteristics, secondary and tertiary structure, stability and ligand-receptor interactions.pET32a and pET28a vectors containing synthetic nisK and nisR genes were transformed into E. coli followed by IPTG induction. SDS-PAGE and western blotting methods were applied to confirm the presence and identity of the amplified proteins. Following purification, the proteins were dialyzed and then prepared for activity assay. The CAI index showed that the genes was compatible with the E. coli host and that the proteins have effective expression. Also, the mRNA prediction results suggest that there is enough mRNA stability for efficient translation in the new host. NisK and NisR recombinant proteins were expressed in E. coli with half - lives of around 10h and were confirmed with molecular weights of 27kDa and 69kDa, respectively, by SDS-PAGE and western blotting. The secondary structure of the recombinant proteins as predicted by circular dichroism spectroscopy was similar to the in silico protein structures. Activity assay of recombinant NisK was performed by measuring the amount of consumed ATP according to the light produced by luciferase. Because NisK and NisR have a direct impact on each other, they have an essential role in increasing the production of nisin and they can be used in different research fields. Our results demonstrated that recombinant proteins NisK and NisR preserved their structure and function after expression.

Purification and characterization of turanose, a sucrose isomer and its anti-inflammatory effects in dextran sulfate sodium (DSS)-induced colitis model

Abstract The prevalence of inflammatory bowel disease (IBD) is increasing worldwide, and the key risk factor is high sugar diet. The objective of this study was to investigate anti-inflammatory effects of turanose on IBD in a dextran sulfate sodium (DSS)-induced colitis mouse model. An amylosucrase from Neisseria sicca (NsiAS) was newly identified and successfully expressed. This enzyme exhibited optimal activity at 45 °C and pH 8.5. By utilizing this recombinant enzyme expressed in the E. coli host, turanose yield maximally reached 47% from 2.0 M sucrose. Colitis was induced with two 5-d cycles of 2.5% DSS in drinking water with a 10-d inter-cycle interval. Mice were fed AIN93G diet with 25% and 50% turanose replacement. Improved disease activity index scores, colon length, histopathological features, and myeloperoxidase activity were observed in the turanose supplemented group. Meanwhile, decreases in micro RNA (miR)-21 expression, histone acetylation, expression of pro-inflammatory cytokines, and phosphorylated ERK and STAT3 were observed in colon tissues. Notably, miR-21 and histone acetylation levels were found to positively correlated with expression of tumor necrosis factor α. In conclusion, turanose can attenuate colitis via regulatory effects on miRNA-21 expression and histone acetylation-related proinflammatory mediators. These findings also support further exploration of turanose as a potential therapeutic sugar substitute for patients with IBD.

Expression, Purification, and in vivo Evaluation of GFP-Fused M Cell Targeting Receptor Binding Domain of Protein FimH.

The number of oral vaccines is still limited due to many difficulties suffered in the intestinal environment, such as mucosal clearance, vast area, harsh conditions, deteriorative enzymes, impermeability, tolerance, etc. Numerous strategies have focused on directing antigen to the receptors of M cells, which is the main gateway to acquire and initiate specific responses to antigens in intestine. FimHrb is a receptor binding domain of type 1 of fimbriae from E. coli and Salmonella that can bind to GP2 receptor expressed exclusively on M cells. In this study, we evaluated the potential of FimHrb for oral vaccine development via its ability to adhere M cells. The coding gene of FimHrb fused Green Fluorescent Protein (GFP) was cloned and expressed intracellularly in E. coli host strain. The recombinant protein FimHrb-GFP was then purified by IMAC method through 6x His tag designed downstream of GFP. Finally, the purified protein was monitored its binding on murine M cells in Payer Patch region. Following the methods mentioned above, the coding gene FimHrb-GFP was successfully cloned into vector pET22b and intracellularly expressed in soluble form at low temperature induction. The purity and the recovered yield of this protein were 90% and 20%, respectively. After that, the adhesion of FimHrb-GFP was monitored in murine small intestine, which showed that the protein bound to Peyer Patch region and did not restrict on M cells. With the present data, we revealed a candidate protein FimHrb targeted receptor on M cells for oral vaccine development and other factors in E. coli would supplement FimH to provide the specific invasion of these bacteria via M cells.

Characterization of MenA (isoprenyl diphosphate:1,4-dihydroxy-2-naphthoate isoprenyltransferase) from Mycobacterium tuberculosis.

The menaquinone biosynthetic pathway presents a promising drug target against Mycobacterium tuberculosis and potentially other Gram-positive pathogens. In the present study, the essentiality, steady state kinetics of MenA from M. tuberculosis and the mechanism of MenA inhibition by Ro 48–8071 were characterized. MenA [isoprenyl diphosphate:1,4-dihydroxy-2-naphthoate (DHNA) isoprenyltransferase] catalyzes a critical reaction in menaquinone biosynthesis that involves the conversion of cytosolic DHNA, to membrane bound demethylmenaquinone by transferring a hydrophobic 45-carbon isoprenoid chain (in the case of mycobacteria) to the ring nucleus of DHNA. Rv0534c previously identified as the gene encoding MenA in M. tuberculosis complemented a menA deletion in E. coli and an E. coli host expressing Rv0534c exhibited an eight-fold increase in MenA specific activity over the control strain harboring empty vector under similar assay conditions. Expression of Rv0534c is essential for mycobacterial survival and the native enzyme from M. tuberculosis H37Rv was characterized using membrane preparations as it was not possible to solubilize and purify the recombinant enzyme. The enzyme is absolutely dependent on the presence of a divalent cation for optimal activity with Mg+2 being the most effective and is active over a wide pH range, with pH 8.5 being optimal. The apparent Km values for DHNA and farnesyl diphosphate were found to be 8.2 and 4.3 μM, respectively. Ro 48–8071, a compound previously reported to inhibit mycobacterial MenA activity, is non-competitive with regard to DHNA and competitive with regard to the isoprenyldiphosphate substrate.

Does the MS2 L protein work with E. coli host factors?

MS2 is a small single stranded RNA bacteriophage that escapes its infected Escherichia coli host through an L protein‐driven lysis mechanism. The L protein consists of 75 amino acids, with a hydrophilic N‐terminal that contains mainly basic residues and a hydrophobic C‐terminal end. Prior studies have shown that the L protein is necessary for the cell to lyse, however it is not known if L alone is sufficient for cell lysis. In our laboratory we have shown that expression of the L protein in the absence of other viral proteins causes E. coli lysis within 40 minutes of expression. Further, the actual mechanism for lysis is not completely understood and potentially requires a protein‐protein interaction with E. Coli host factors. Recent studies by Chamakura et. al [Journal of Bacteriology, 12, 199 (2017)] using a genetic screen identified DnaJ, a chaperone protein, as a host factor involved in the lysis mechanism. However, this study did not describe a direct protein‐protein interaction between L and DnaJ. Thus, the question remains whether DnaJ along with MS2 L is necessary and sufficient for lysis, or are there additional host proteins involved? In order to answer this question and investigate host protein interactions using a biochemical approach, a hemagglutinin epitope tag (HA) will be inserted into a plasmid containing the MS2 L gene and an arabinose‐inducible promoter. Once transformed into E. coli, the HA tagged L protein can be detected using a Western Blot and purified by immunoprecipitation. Using these methods, it is expected that any host protein that interacts with the L protein will be co‐immunoprecipitated. Future studies will use mass spectrometry to identify these co‐immunoprecipitated proteins.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comparison of the Activities among Three SUL Genes Present in Uropathogenic Escherichia Coli

The three plasmid borne alternative dihydropteroate synthetase (DHPS) genes namely sul1, sul2 and sul3 genes were heterologous in amino acid sequence and have about 40 – 50% identity. However, they have same DHPS activity with disc diffusion zone size of 6 mm with suphamethoxazole disc in our previous study. Sul1, sul2 and sul3 genes were observed in sulphamethoxazole resistant uropathogenic Escherichia coli (UPEC). In this study, all the three genes were cloned into E. coli host and minimum inhibitory concentration (MIC) was investigated for each sul gene to compare the activities of sul genes. The MIC values of E.coli containing sul2, sul1 and sul3 genes inserted recombinant plasmid were observed to have 18.5 mg/ml, 18 mg/ml and 17.5 mg/ml respectively as mean value of five experimental results. Although comparable MICs were obtained as a result, the MIC value was highest in E. coli carrying sul2 gene indicating that this DHPS enzyme activity of sul2 was strongest among three sul genes.

Type II flavoprotein monooxygenase PsFMO_A from the bacterium Pimelobacter sp. Bb-B catalyzes enantioselective Baeyer-Villiger oxidations with a relaxed cofactor specificity

Microbial consortia, which degrade branched, long-chain hydrocarbons, can be regarded as a promising source of novel enzymes for the stereo- and regio-selective oxyfunctionalization of hydrocarbons. The hydrocarbon-degrading bacterium Pimelobacter sp. Bb-B was isolated from the consortium associated with the colonial hydrocarbon-excreting microalga Botryococcus braunii. Three new type II flavoprotein monooxygenases (type II FMOs) from this bacterium have been made available in recombinant form through cloning and overexpression in an E. coli host organism. These enzymes (PsFMO_A-C) were characterized in terms of their capability of catalyzing Baeyer-Villiger oxidations with distinct substrates. The highest activity was detected when utilizing camphor and bicyclo[3.2.0]hept-2-en-6-one as substrate in combination with PsFMO_A as the most promising enzyme. Furthermore, synthetic biotransformations with 5 mM of the substrate bicyclo[3.2.0]hept-2-en-6-one, formate and formate dehydrogenase for in situ-cofactor recycling were conducted with this enzyme, leading to a substrate consumption of 85% after 66 h and excellent enantioselectivity of 99% ee for the (1R,5S)-enantiomer. Additionally, an alternative in situ-cofactor recycling based on the use of microalgae for in situ-production of formate from carbon dioxide, water and light together with a formate dehydrogenase was combined successfully with the enzyme PsFMO_A, leading to a substrate consumption of 94% and an enantioselectivity of >99% ee for the so-called “normal lactone”-enantiomer with the absolute configuration 1R,5S.

Quorum sensing in Pseudomonas aeruginosa mediated by RhlR is regulated by a small RNA PhrD

Pseudomonas aeruginosa is a highly invasive human pathogen in spite of the absence of classical host specific virulence factors. Virulence factors regulated by quorum sensing (QS) in P. aeruginosa cause acute infections to shift to chronic diseases. Several small regulatory RNAs (sRNAs) mediate fine-tuning of bacterial responses to environmental signals and regulate quorum sensing. In this study, we show that the quorum sensing regulator RhlR is positively influenced upon over expression of the Hfq dependent small RNA PhrD in Pseudomonas. RhlR transcripts starting from two of the four different promoters have same sequence predicted to base pair with PhrD. Over expression of PhrD increased RhlR transcript levels and production of the biosurfactant rhamnolipid and the redox active pyocyanin pigment. A rhlR::lacZ translational fusion from one of the four promoters showed 2.5-fold higher expression and, a 9-fold increase in overall rhlR transcription was seen in the wild type when compared to the isogenic phrD disruption mutant. Expression, in an E. coli host background, of a rhlR::lacZ fusion in comparison to a construct that harboured a scrambled interaction region resulted in a 10-fold increase under phrD over expression. The interaction of RhlR-5′UTR with PhrD in E. coli indicated that this regulation could function without the involvement of any Pseudomonas specific proteins. Overall, this study demonstrates that PhrD has a positive effect on RhlR and its associated physiology in P. aeruginosa.

Rational engineering of a malate dehydrogenase for microbial production of 2,4-dihydroxybutyric acid via homoserine pathway.

A synthetic pathway for the production of 2,4-dihydroxybutyric acid from homoserine (HMS), composed of two consecutive enzymatic reaction steps has been recently reported. An important step in this pathway consists in the reduction in 2-keto-4-hydroxybutyrate (OHB) into (l)-dihydroxybutyrate (DHB), by an enzyme with OHB reductase activity. In the present study, we used a rational approach to engineer an OHB reductase by using the cytosolic (l)-malate dehydrogenase from Escherichia coli (Ec-Mdh) as the template enzyme. Structural analysis of (l)-malate dehydrogenase and (l)-lactate dehydrogenase enzymes acting on sterically cognate substrates revealed key residues in the substrate and co-substrate-binding sites responsible for substrate discrimination. Accordingly, amino acid changes were introduced in a stepwise manner into these regions of the protein. This rational engineering led to the production of an Ec-Mdh-5E variant (I12V/R81A/M85E/G179D/D86S) with a turnover number (kcat) on OHB that was increased by more than 2000-fold (from 0.03 up to 65.0 s-1), which turned out to be 7-fold higher than that on its natural substrate oxaloacetate. Further kinetic analysis revealed the engineered enzyme to possess comparable catalytic efficiencies (kcat/Km) between natural and synthetic OHB substrates (84 and 31 s-1 mM-1, respectively). Shake-flask cultivation of a HMS-overproducing E. coli strain expressing this improved OHB reductase together with a transaminase encoded by aspC able to convert HMS to OHB resulted in 89% increased DHB production as compared with our previous report using a E. coli host strain expressing an OHB reductase derived from the lactate dehydrogenase A of Lactococcus lactis.

A comparative study of the bispecific monoclonal antibody, blinatumomab expression in CHO cells and E. coli

The “bispecifics” market improved over the past decade due to the development of many technological platforms including bispecific T cell engagers (BiTEs). The approval of blinatumomab, the most advanced bispecific T-cell engager (BiTE) in clinical trials, can be a significant milestone in the development of bispecific antibodies. Both Chinese hamster ovary (CHO) cells and E. coli strain are considered as the most widely used hosts for the large-scale production of therapeutic monoclonal antibodies. Since both of the economic and qualitative aspects of protein production are important in industry, selection of a suitable protein expression system is very critical. The BsAb gene was cloned into the expression vectors FC550A-1, pcDNA3.1 (+), and PET22b and 6 × His-tagged BsAb then purified on a Ni-NTA chromatography column. Both SDS–PAGE and Western blotting analysis of the purified protein demonstrated that blinatumomab was successfully expressed as a 55 kDa in both expression systems. The antigen-binding properties of blinatumomab were compared in the mammalian system versus Escherichia coli. The results showed that the purified antibody from a mammalian expression system has better binding activity than the one from E. coli host.

Emergence of the colistin resistance gene mcr-1 and its variant in several uncommon species of Enterobacteriaceae from commercial poultry farm surrounding environments

The colistin resistance gene mcr-1 has been detected in multiple members of Enterobacteriaceae family. Here, we report the emergence of mcr-1 in Providencia alcalifaciens and a mcr-1 variant, named mcr-1.3, in Raoultella planticola. Both of the mcr-1-carrying plasmids in these two isolates belong to IncI2 type of plasmids, but they are different in sizes and genetic characteristics. We also detected the mcr-1 gene in one Enterobacter cloacae isolate, however, the mcr-1-carrying plasmid is distinct from the previous reports. Conjugation assay showed that mcr-1-carrying plasmids in P. alcalifaciens and E. cloacae were successfully transferred into recipient E. coli strains. It is worth noting that the transferability of mcr-1-carrying plasmid from E. cloacae was enhanced once it entered into E. coli hosts, which might further accelerate the dissemination of mcr-1 among Enterobacteriaceae. These findings further expand our knowledge of the mcr-1-carrying species in Enterobacteriaceae.

Physicochemical characterization of E. coli-derived human serum albumin and its comparison with the human plasma counterpart reveals it as a promising biosimilar

Human serum albumin one of the most demanded proteins possess an array of clinical and biotechnological applications. Currently, the prime source for HSA production is the human blood which possesses the risk of pathogen contamination and is limited. Thus, there exists an indispensable need to promote non-animal derived HSA production. In the present work, we have exploited the opportunity and promoted the preparation of pathogen-free rHSA from the E. coli host which is blessed with numerous advantages like scalability, cost-effectiveness etc. Upon overcoming the difficulties to produce functional rHSA in E. coli, through engineering the biological system of protein folding in the cell, the E. coli-derived rHSA has been purified to homogeneity. Its detailed physicochemical characterization has been performed, by monitoring its conformational properties, secondary and tertiary structure elements, surface properties, ligand binding properties, stability issues etc. These parameters of the recombinant protein have been compared with the naturally occurring protein from the human source. The outcome of the comparison reveals that the recombinant protein resembles exactly the same as the natural one. Hence, we propose and promote that the E. coli-derived rHSA is an ideal biosimilar for human blood plasma-derived serum albumin.

Compartmentalized Self-Replication for Evolution of a DNA Polymerase.

Compartmentalized self-replication (CSR) is an emulsion PCR-based method for the selection of DNA polymerases. E. coli host cells expressing a library of DNA polymerases are emulsified so that no more than a single cell is present in a single emulsion droplet. In a subsequent emulsion PCR step, the DNA polymerase protein, as well as the plasmid encoding it are released into the emulsion droplet and the genes that created the most active or abundant polymerase variants are exponentially amplified and can be passed to the next round of CSR. CSR is a powerful method for engineering of polymerases since it allows selection under a variety of conditions, including the use of non-standard substrates. In this unit, we provide a step-by-step procedure for the selection of polymerases, using as an example the selection of reverse transcriptase activity starting from a library of Thermococcus kodakaraensis (KOD) DNA polymerase variants. © 2018 by John Wiley & Sons, Inc.

In silico Analysis of Several Signal Peptides for the Excretory Production of Reteplase in Escherichia coli

Background: Solubility of recombinant therapeutic enzymes is one of the major purpose their production in bacterial host The factors such as metabolic system, a cellular stress response of the bacterial host and formation of inclusion bodies cause undesirable expression results. One method to overcome this problem is secretion of recombinant protein into the extracellular growth medium by selection of proper signal peptides. Reteplase (Retavase) is a serine protease simulating endogenous tissue plasminogen activator (t-PA), fibrin-specific plasminogen activator, playing role in thrombolysis and fibrinolysis (clot lysis) by converting plasminogen to plasmin. Objective: The aim of this study was to evaluate several signal peptides in order to selection the proper one which suitable for excretory production of the reteplase. Method: The presence and cleavage site location in signal peptides was predicted by SignalP 4.1. The prediction of secretory pathway of signal peptides was performed using high accurate database. Several critical features of the signal peptide were evaluated applying various online web-servers such as ProtParam and Solpro with high sensitivity and specificity. Finally, the selected signal peptides were compared and the one which was appropriate candidate for high yields production of secretory reteplase in the E. coli host was defined. Results: Among total 30 signal peptides, 6 of them, CHIS_BACSU, MPT53_MYCTU, PET_ECOLX, PIC_ECOL6, PIC_ECLOX, TSH_ECOLX were omitted. CPLR_DESHA, AGAR_ALTAT, CDGT_BACS2, PAG_BACAN could be considered as proper candidate for efficient extracellular production of reteplase in E. coli. Conclusion: Theoretical analysis and evaluation of signal peptides by using computational tools can be acceptably carried out, but these results must be established via practical effort. Keywords: E. coli host, bioinformatics, secretion, physicochemical properties, solubility, signal peptides.

Revisiting Escherichia coli as microbial factory for enhanced production of human serum albumin

BackgroundHuman serum albumin (HSA)—one of the most demanded therapeutic proteins with immense biotechnological applications—is a large multidomain protein containing 17 disulfide bonds. The current source of HSA is human blood plasma which is a limited and unsafe source. Thus, there exists an indispensable need to promote non-animal derived recombinant HSA (rHSA) production. Escherichia coli is one of the most convenient hosts which had contributed to the production of more than 30% of the FDA approved recombinant pharmaceuticals. It grows rapidly and reaches high cell density using inexpensive and simple subst-rates. E. coli derived recombinant products have more economic potential as fermentation processes are cheaper compared to the other expression hosts. The major bottleneck in exploiting E. coli as a host for a disulfide-rich multidomain protein is the formation of aggregates of overexpressed protein. The majority of the expressed HSA forms inclusion bodies (more than 90% of the total expressed rHSA) in the E. coli cytosol. Recovery of functional rHSA from inclusion bodies is not preferred because it is difficult to obtain a large multidomain disulfide bond rich protein like rHSA in its functional native form. Purification is tedious, time-consuming, laborious and expensive. Because of such limitations, the E. coli host system was neglected for rHSA production for the past few decades despite its numerous advantages.ResultsIn the present work, we have exploited the capabilities of E. coli as a host for the enhanced functional production of rHSA (~ 60% of the total expressed rHSA in the soluble fraction). Parameters like intracellular environment, temperature, induction type, duration of induction, cell lysis conditions etc. which play an important role in enhancing the level of production of the desired protein in its native form in vivo have been optimized. We have studied the effect of assistance of different types of exogenously employed chaperone systems on the functional expression of rHSA in the E. coli host system. Different aspects of cell growth parameters during the production of rHSA in presence and absence of molecular chaperones in E. coli have also been studied.ConclusionIn the present case, we have filled in the gap in the literature by exploiting the E. coli host system, which is fast-growing and scalable at the low cost of fermentation, as a microbial factory for the enhancement of functional production of rHSA, a crucial protein for therapeutic and biotechnological applications.

Ultra-High Efficient Colony PCR for High Throughput Screening of Bacterial Genes.

Current colony PCR methods are not suitable for screening genes encoded in genomic DNA and are limited to E. coli host strains. Here, we describe an ultra-high efficient colony PCR method for high throughput screening of bacterial genes embedded in the genomic DNA of any bacterial species. This new technique expands colony PCR method to several hosts as well as offers a rapid, less expensive and reliable bacterial genomic DNA extraction.

Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli

Codon usage is one of the factors influencing recombinant protein expression. We were interested in the codon usage of an antibody Fab fragment gene exhibiting extreme toxicity in the E. coli host. The toxic synthetic human Fab gene contained domains optimized by the “one amino acid-one codon” method. We redesigned five segments of the Fab gene with a “codon harmonization” method described by Angov et al. and studied the effects of these changes on cell viability, Fab yield and display on filamentous phage using different vectors and bacterial strains. The harmonization considerably reduced toxicity, increased Fab expression from negligible levels to 10 mg/l, and restored the display on phage. Testing the impact of the individual redesigned segments revealed that the most significant effects were conferred by changes in the constant domain of the light chain. For some of the Fab gene variants, we also observed striking differences in protein yields when cloned from a chloramphenicol resistant vector into an identical vector, except with ampicillin resistance. In conclusion, our results show that the expression of a heterodimeric secretory protein can be improved by harmonizing selected DNA segments by synonymous codons and reveal additional complexity involved in heterologous protein expression.