Nickel Affinity Chromatography Research Articles

Cloning and expression of in silico modeled protein enriched with branched chain amino acids in Pichia pastoris

We have earlier in silico designed the 3-dimensional structure of a protein enriched with branched chain amino acids (BCAA, 56.4%), having only α-helical coiled-coil structure. Here, homology modeling was used to improve the in silico designed protein model. The secondary and tertiary structures of improved protein model were predicted, and validated using various online bioinformatics tools. The amino acid sequence of the final predicted Protein Model-51 was EQLTKLEIVIRVLKLLKLIGGLVSLVEWVLTALVTLLGDKVLDDILTDVIMLVKKIL DKVIGIVYVLAILALILSEVLDILWLLEKLVEILEGHHHHHH. The amino acid sequence of the protein model was reverse translated to DNA sequence and codons were optimized using codon optimization tool. The chemically synthesized BCAA51 gene was cloned to pPICZαC vector, and transformed into DH5α E. coli strain. After successful transformation, the protein was expressed in P. pastoris system by inducing with 0.5% methanol, every 24 h for up to 144 h. The expressed protein was purified by His Select Nickel affinity chromatography with an yield of 1.412 mg/L. The recombinant protein was confirmed by SDS-PAGE and western blot analysis, which showed a clear band at the expected molecular weight of ~11 kDa. Thus, here we have shown that the in silico designed protein is successfully cloned and expressed in P. pastoris.

Production of a kinesin-related recombinant protein (Lbk39) from Leishmania braziliensis by Leishmania tarentolae promastigotes and its application in the serodiagnosis of leishmaniasis

The leishmaniases are multifactorial zoonotic diseases requiring a multidisciplinary One Health approach for diagnosis and control. For leishmaniasis diagnosis, here we describe production of a new recombinant protein based on a kinesin-related gene of Leishmania braziliensis (Lbk39), which shows 59% amino acid identity to the L. infantum homologue. The Lbk39 gene was synthesized, inserted into the pLEXSY-sat2 vector and transfected into L. tarentolae cells by electroporation. Culturing was carried out, and the secreted recombinant protein with a C-terminal histidine tag purified using nickel affinity chromatography on the culture supernatant, yielding a final product at 0.4 mg/mL. An indirect enzyme linked immunosorbent assay (ELISA) was standardised using sera from 74 Brazilian patients with cutaneous leishmaniasis and 11 with visceral leishmaniasis. Optimal ELISA conditions were established for the Lbk39 antigen in comparison with a crude extract from L. braziliensis. The sensitivity, specificity analysis and receiver operating characteristic (ROC) curve were determined with a significance level of 5%. The ROC curve showed a good accuracy with an area under curve (AUC) = 0.967, p < 0.001 (0.941–0.993) for CL patients and an AUC = 100 (100-100) for VL patients. The values of sensitivity and specificity were 88 and 98% for CL and 100 and 100% for VL, respectively. The study showed good production and expression of the target protein and has generated a potential new antigen for the diagnosis of leishmaniasis.

Preparation and Purification of Recombinant Protein Fragment OmpA240-356 from Acinetobacter baumannii as a Novel Epitope for Vaccination

Acinetobacter baumannii has gained attention for years as a significant clinical problem due to the increase of antibiotic-resistant strain. Indeed, A. baumannii OmpA is one of the highly conserved membrane proteins among Gram-negative bacteria that has multiple roles in interacting with the host during infection, thereby representing an effective target for the development of novel an-tibacterial or vaccination therapies. Nowadays, finding suitable epitope-specific antigens inside the conserved proteins such as OmpA is a promising method for successful vaccination programs. Therefore, in the present study, the coding sequence of the 240 to 356 amino acid residues of A. baumannii OmpA (AbOmpA240-356) was cloned into the vector pET-28a and purified using nickel affinity chromatography. In addition, the anti-His tag antibody is used to validate its production. This system of protein expression and purification may be useful for further characterization of AbOmpA240-356 protein fraction. Therefore, this study can lead to the introduction of suitable candidates for the development of an effective vaccine based on OmpA against this bacterium for further analysis. © 2019, Archives of Clinical Infectious Diseases.

Development of a human scFv antibody targeting the lethal Iranian cobra (Naja oxiana) snake venom

Snakebite is one of the health concerns worldwide. Naja oxiana is one of the venomous snakes with a high mortality rate. Anti-serum therapy is the only treatment of the victims. However, in some cases, antiserum injection leads to some side effects in host like serum sickness and anaphylactic shock. It is crucial to develop a neutralizing agent with low side effects. The human antibody library (non-immunized library) was used to isolate specific antibodies against N.oxiana venom components. Four rounds of biopanning were performed to enrich scFv-displaying phages against the venom of N. oxiana. Enrichment of scFv-displaying phages against N. oxiana venom was analyzed by polyclonal Enzyme-Linked Immunosorbent Assay (ELISA). Specific antibody fragments against N. oxiana venom were selected through monoclonal ELISA, and were expressed in E. coli bacterial cells. Purification of the selected clones was performed by using nickel affinity chromatography. Neutralization and protective capacity of specific antibody fragments were analyzed in C57BL/6 mice (i.v. injection). Results of biopanning and polyclonal ELISA demonstrate a successful enrichment process. Five specific antibody fragments with the highest signal in monoclonal ELISA were selected, expressed, and purified. The purity of expressed antibody fragments was monitored by SDS-PAGE and Western blot. The selected antibody fragments were able to neutralize two LD50 of N. oxiana venom and protected all mice when injected 15 min post-envenomation. The data indicate that such selected antibodies are promising tools for further studies and in the development of novel protective agents against N. oxiana venom.

Development of a mono-specific anti-VEGF bivalent nanobody with extended plasma half-life for treatment of pathologic neovascularization.

Vascular endothelial growth factor (VEGF) plays a crucial role in angiogenesis within solid cancers. Thus, targeting VEGF might be part of a feasible therapy for treating pathological neovascularization, and nanobodies - derived from heavy chain-only antibodies occurring within Camelidae - are a novel class of nanometer-sized antibodies possessing unique properties that could be developed into a promising therapeutic. However, nanobodies have a very short half-life in vivo due to their small size. Development of a bivalent nanobody is one way to remediate the half-life problem of nanobodies. Two identical anti-VEGF nanobodies were connected using the hinge region of llama IgG2c. The recombinant plasmid (pHEN6c-bivalent nanobody) was transformed into E.coli WK6 cells and expression of the bivalent nanobody construct was induced with 1mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). Recombinant bivalent nanobody was purified using nickel affinity chromatography and its activity on human endothelial cells was assessed using 3-(4,5-Dimethylthiazol-2-yr)-2,5-diphenyltetrazolium bromide (MTT), tube formation, and cell migration assays. The pharmacokinetic study was performed after intravenous (i.v.) injection of recombinant bivalent nanobody into six-week-old C57BL/6 mice. Recombinant bivalent nanobody performed significantly better than monovalent nanobody in inhibiting proliferation, tube formation, and migration of human endothelial cells. Pharmacokinetic results showed a 1.8-fold longer half-life of bivalent nanobody in comparison with the monovalent nanobody. These results underscore the potential of recombinant anti-VEGF bivalent nanobody as a promising tool for development of a novel therapeutic with an extended plasma half-life for VEGF-related diseases.

The Arabian camel, Camelus dromedarius interferon epsilon: Functional expression, in vitro refolding, purification and cytotoxicity on breast cancer cell lines.

The current study highlights, for the first time, cloning, overexpression and purification of the novel interferon epsilon (IFNƐ), from the Arabian camel Camelus dromedaries. The study then assesses the cytotoxicity of IFNε against two human breast cancer cell lines MDA-MB-231 and MCF-7. Full-length cDNA encoding interferon epsilon (IFNε) was isolated and cloned from the liver of the Arabian camel, C. dromedarius using reverse transcription-polymerase chain reaction. The sequence analysis of the camel IFNε cDNA showed a 582-bp open reading frame encoding a protein of 193 amino acids with an estimated molecular weight of 21.230 kDa. A BLAST search analysis revealed that the C. dromedarius IFNε shared high sequence identity with the IFN genes of other species, such as Camelus ferus, Vicugna pacos, and Homo sapiens. Expression of the camel IFNε cDNA in Escherichia coli gave a fusion protein band of 24.97 kDa after induction with either isopropyl β-D-1-thiogalactopyranoside or lactose for 5 h. Recombinant IFNε protein was overexpressed in the form of inclusion bodies that were easily solubilized and refolded using SDS and KCl. The solubilized inclusion bodies were purified to apparent homogeneity using nickel affinity chromatography. We examined the effect of IFNε on two breast cancer cell lines MDA-MB-231 and MCF-7. In both cell lines, IFNε inhibited cell survival in a dose dependent manner as observed by MTT assay, morphological changes and apoptosis assay. Caspase-3 expression level was found to be increased in MDA-MB-231 treated cells as compared to untreated cells.

Prokaryotic expression and transmembrane transfer of fusion protein TAT-RIG-I-GFP

We studied the construction of fusion protein TAT-RIG-I-GFP prokaryotic expression vector and verified the function of TAT in transmembrane delivery. First, four pairs of specific primers were designed, and the RIG-I gene of Mallard Duck (Anas platyrhynchos) was cloned. Then, the pET-TAT-RIG-I-GFP and pET-RIG-I-GFP prokaryotic expression vectors were constructed. Meanwhile, they were converted to E. coli BL21 (DE3), which were induced to be expressed after culture. After the purification of His-60 nickel affinity chromatography column and the identification of SDS-PAGE, the purified TAT-RIG-I-GFP and RIG-I-GFP proteins were incubated to DF-1 cells. Finally, fluorescence microscopy was used to observe whether the corresponding fluorescence was produced in DF-1 cells. The results showed that pET-TAT-RIG-I-GFP fusion with TAT showed obvious green fluorescence in DF-1 cells. However, the pET-RIG-I-GFP without TAT cannot display green fluorescence. This shows that TAT-fused protein have successfully delivered DF-1 cells and play a key role in transmembrane delivery. In conclusion, these results provide a solid material basis for further study of antiviral drugs in poultry.

Characterization of an Uncinocarpus reesii-expressed recombinant tube precipitin antigen of Coccidioides posadasii for serodiagnosis.

Early and accurate diagnosis of coccidioidomycosis, also known as Valley fever, is critical for appropriate disease treatment and management. Current serodiagnosis is based on the detection of patient serum antibodies that react with tube precipitin (TP) and complement fixation (CF) antigens of Coccidioides. IgM is the first class of antibodies produced by hosts in response to coccidioidal insults. The highly glycosylated β-glucosidase 2 (BGL2) is a major active component of the TP antigen that stimulates IgM antibody responses during early Coccidioides infection. The predominant IgM epitope on BGL2 is a unique 3-O-methyl-mannose moiety that is not produced by commonly used protein expression systems. We genetically engineered and expressed a recombinant BGL2 (rBGL2ur), derived from Coccidioides, in non-pathogenic Uncinocarpus reesii, a fungus phylogenetically related to the Coccidioides pathogen. The rBGL2ur protein was purified from the culture medium of transformed U. reesii by nickel affinity chromatography, and the presence of 3-O-methyl mannose was demonstrated by gas chromatography. Seroreactivity of the purified rBGL2ur protein was tested by enzyme-linked immunosorbent assays using sera from 90 patients with coccidioidomycosis and 134 control individuals. The sensitivity and specificity of the assay with rBGL2ur were 78.8% and 87.3%, respectively. These results were comparable to those obtained using a proprietary MiraVista Diagnostic (MVD) IgM (63.3% sensitivity; 96.3% specificity), but significantly better than the ID-TP assay using non-concentrated patient sera (33.3% sensitivity; 100% specificity). Expression of rBGL2ur in U. reesii retains its antigenicity for coccidioidomycosis serodiagnosis and greatly reduces biosafety concerns for antigen production, as Coccidioides spp. are biological safety level 3 agents.

Expression, purification and characterisation of Trypanosoma congolense metacaspase 5 (TcoMCA5) - a potential drug target for animal African trypanosomiasis

The metacaspases (MCAs) are attractive drug targets for the treatment of African trypanosomiasis as they are not found in the metazoan kingdom and their action has been implicated in cell cycle and cell death pathways in kinetoplastid parasites. Here we report the biochemical characterisation of MCA5 from T. congolense. Upon recombinant expression in E. coli, autoprocessing is evident, and MCA5 further autoprocesses when purified using nickel affinity chromatography, which we term nickel-induced over autoprocessing. When both the catalytic His and Cys residues were mutated (TcoMCA5H147A/C202G), no nickel-induced over autoprocessing was observed and was enzymatically active, suggesting the existence of a secondary catalytic Cys residue, Cys81. Immunoaffinity purification of native TcoMCA5 from the total parasite proteins was achieved using chicken anti-TcoMCA5 IgY antibodies. The full length native TcoMCA5 and the autoprocessed products of recombinant TcoMCA5H147A/C202G were shown to possess gelatinolytic activity, the first report for that of a MCA. Both the native and recombinant enzyme were calcium independent, had a preference for Arg over Lys at the P1 site and were active over a pH range between 6.5 and 9. Partial inhibition (23%) of enzymatic activity was only achieved with leupeptin and antipain. These findings are the first step in the biochemical characterisation of the single copy MCAs from animal infective trypanosomes towards the design of novel trypanocides.

The development of an indirect ELISA for the detection of goose parvovirus antibodies using specific VP3 subunits as the coating antigen

BackgroundIn Poland, the leader in goose production in Europe, goose parovirus infection, or Derzsy’s disease (DD), must be reported to the veterinary administration due to the serious economic and epizootic threat to waterfowl production. Prophylactic treatment for DD includes attenuated live or inactivated vaccines. Moreover, the control of DD includes the monitoring of maternal derived antibody (MDA) levels in the offspring and antibody titers in the parent flock after vaccination. The aim of this study was to develop an ELISA for the detection of goose parvovirus (GPV) antibodies.ResultsTwo recombinant protein fragments derived from VP3 (viral protein 3) GPV, namely VP3ep6 and VP3ep4–6 with a mass of 20.9 and 32.3 kDa, respectively, were produced using an Escherichia coli expression system. These proteins were purified by one-step nickel-affinity chromatography, which yielded protein preparations with a purity above 95%. These recombinant proteins were useful in the detection of serum anti-GPV antibodies, and this was confirmed by Western blotting. However, recombinant VP3ep4–6 protein showed a greater ability to correctly identify sera from infected geese. In the next stage of the project, a pool of 166 goose sera samples, previously examined by a virus neutralization test (VN), was tested. For further studies, one recombinant protein (VP3ep4–6) was selected for optimization of the test conditions. After optimization, the newly developed ELISA was compared to other serological tests, and demonstrated high sensitivity and specificity.ConclusionIn conclusion, the VP3ep4–6 ELISA method described here can be used for the detection of antibodies to GPV in serum.

ATPase activity of human binding immunoglobulin protein (BiP) variants is enhanced by signal sequence and physiological concentrations of Mn2.

B‐cell immunoglobulin binding protein (BiP) is an essential endoplasmic reticulum (ER) chaperone normally found in the ER lumen. However, BiP also has other extracellular and intracellular functions. As it is unclear whether peripheral BiP has a signal and/or ER retention sequence, here we produced and biochemically characterised four variants of BiP. The variants differed depending on the presence or the absence of signal and ER retention peptides. Proteins were purified using nickel affinity chromatography, and variant size and quality were confirmed using SDS/PAGE gels. The thermal denaturing temperature of these proteins was found to be 46–47 °C. In addition, we characterised nucleotide binding properties in the absence and the presence of divalent cations. Interestingly, in the absence of cations, ADP has a higher binding affinity to BiP than ATP. The presence of divalent cations results in a decrease of the K d values of both ADP and ATP, indicating higher affinities of both nucleotides for BiP. ATPase assays were carried out to study the enzyme activity of these variants and to characterise the kinetic parameters of BiP variants. Variants with the signal sequence had higher specific activities than those without. Both Mg2+ and Mn2+ efficiently stimulated the ATPase activity of these variants at low micromolar concentrations, whereas calcium failed to stimulate BiP ATPase. Our novel findings indicate the potential functionality of BiP variants that retain a signal sequence, and also reveal the effect of physiological concentrations of cations on the nucleotide binding properties and enzyme activities of all variants.

Enhanced Biocatalytic Activity of Recombinant Lipase Immobilized on Gold Nanoparticles.

Bacterial lipases especially Pseudomonas lipases are extensively used for different biotechnological applications. With the better understanding and progressive needs for improving its activity in accordance with the growing market demand, we aimed in this study to improve the recombinant production and biocatalytic activity of lipases via surface conjugation on gold nanoparticles. The full length coding sequences of lipase gene (lipA), lipase specific foldase gene (lipf) and dual cassette (lipAf) gene were amplified from the genomic DNA of Pseudomonas aeruginosa PA14 and cloned into the bacterial expression vector pRSET-B. Recombinant lipases were expressed in E. coli BL-21 (DE3) pLysS then purified using nickel affinity chromatography and the protein identity was confirmed using SDS-PAGE and Western blot analysis. The purified recombinant lipases were immobilized through surface conjugation with gold nanoparticles and enzymatic activity was colorimetrically quantified. Here, two single expression plasmid systems pRSET-B-lipA and pRSET-B-lipf and one dual cassette expression plasmid system pRSET-B-lipAf were successfully constructed. The lipolytic activities of recombinant lipases LipA, Lipf and LipAf were 4870, 426 and 6740 IUmg-1, respectively. However, upon immobilization of these recombinant lipases on prepared gold nanoparticles (GNPs), the activities were 7417, 822 and 13035 IUmg-1, for LipA-GNPs, Lipf-GNPs and LipAf-GNPs, respectively. The activities after immobilization have been increased 1.52 and 1.93 -fold for LipA and LipAf, respectively. The lipolytic activity of recombinant lipases in the bioconjugate was significantly increased relative to the free recombinant enzyme where immobilization had made the enzyme attain its optimum performance.

Molecular Characterization and Heterologous Production of the Bacteriocin Peocin, a DNA Starvation/Stationary Phase Protection Protein, from Paenibacillus ehimensis NPUST1.

The production of a bacteriocin-like substance with antimicrobial activity, named peocin, by the probiotic Paenibacillus ehimensis NPUST1 was previously reported by our laboratory. The present study aimed to identify peocin and increase the peocin yield by heterologous expression in Escherichia coli BL21(DE3). Peocin was identified as a DNA starvation/stationary phase protection protein, also called DNA-binding protein from starved cells (Dps), by gel overlay and LC-MS/MS analysis. For mass production of peocin, fed-batch cultivation of E. coli was performed using a pH-stat control system. Purification by simple nickel affinity chromatography and dialysis yielded 45.3 mg of purified peocin from a 20-mL fed-batch culture (49.3% recovery). The biological activity of the purified peocin was confirmed by determination of the MIC and MBC against diverse pathogens. Purified peocin exhibited antimicrobial activity against aquatic, food spoilage, clinical and antibiotic-resistant pathogens. In an in vivo challenge test, zebrafish treated with purified peocin exhibited significantly increased survival rates after A. hydrophila challenge. The present study is the first to show the antimicrobial activity of Dps and provides an efficient strategy for production of bioactive peocin, which will aid the development of peocin as a novel antimicrobial agent with potential applications in diverse industries.

Fusion with an albumin-binding domain improves pharmacokinetics of an αvβ3-integrin binding fibronectin scaffold protein.

Fusion with an albumin-binding domain (ABD) of streptococcal protein G represents a popular approach for half-life extension of small protein therapeutics in the organism. To increase the circulation time of engineered αvβ3-integrin-binding protein (JCL) based on the 10th human fibronectin type III domain (10 Fn3), we have constructed several fusions with ABD with different orientations of the partner proteins and linker length. The recombinant proteins were expressed in Escherichia coli cells and purified by nickel-affinity chromatography. All fusion proteins bound human serum albumin (HSA) in ELISA assay; however, fusions with longer linkers demonstrated better performance. Interaction of ABD-L15 -JCL and JCL-L14 -ABD with HSA was confirmed by analytical size exclusion chromatography and pull-down assays. Surprisingly, the thermal stability of ABD-L15 -JCL was dramatically decreased in comparison with JCL and JCL-L14 -ABD proteins. Pharmacokinetic studies revealed that JCL-L14 -ABD circulated in murine blood about 10 times longer than ABD-L15 -JCL and 960 times longer than JCL. Biodistribution studies of JCL-L14 -ABD in mice revealed its increased level in blood and a decreased accumulation in liver and kidneys in comparison with JCL. Obtained results demonstrate the utility of the fusion with ABD for half-life extension of the binding proteins based on 10 Fn3.

Periplasmic Expression of 4/7 α-Conotoxin TxIA Analogs in E. coli Favors Ribbon Isomer Formation - Suggestion of a Binding Mode at the α7 nAChR.

Peptides derived from animal venoms provide important research tools for biochemical and pharmacological characterization of receptors, ion channels, and transporters. Some venom peptides have been developed into drugs (such as the synthetic ω-conotoxin MVIIA, ziconotide) and several are currently undergoing clinical trials for various clinical indications. Challenges in the development of peptides include their usually limited supply from natural sources, cost-intensive chemical synthesis, and potentially complicated stereoselective disulfide-bond formation in the case of disulfide-rich peptides. In particular, if extended structure–function analysis is performed or incorporation of stable isotopes for NMR studies is required, the comparatively low yields and high costs of synthesized peptides might constitute a limiting factor. Here we investigated the expression of the 4/7 α-conotoxin TxIA, a potent blocker at α3β2 and α7 nicotinic acetylcholine receptors (nAChRs), and three analogs in the form of maltose binding protein fusion proteins in Escherichia coli. Upon purification via nickel affinity chromatography and release of the toxins by protease cleavage, HPLC analysis revealed one major peak with the correct mass for all peptides. The final yield was 1–2 mg of recombinant peptide per liter of bacterial culture. Two-electrode voltage clamp analysis on oocyte-expressed nAChR subtypes demonstrated the functionality of these peptides but also revealed a 30 to 100-fold potency decrease of expressed TxIA compared to chemically synthesized TxIA. NMR spectroscopy analysis of TxIA and two of its analogs confirmed that the decreased activity was due to an alternative disulfide linkage rather than the missing C-terminal amidation, a post-translational modification that is common in α-conotoxins. All peptides preferentially formed in the ribbon conformation rather than the native globular conformation. Interestingly, in the case of the α7 nAChR, but not the α3β2 subtype, the loss of potency could be rescued by an R5D substitution. In conclusion, we demonstrate efficient expression of functional but alternatively folded ribbon TxIA variants in E. coli and provide the first structure–function analysis for a ribbon 4/7-α-conotoxin at α7 and α3β2 nAChRs. Computational analysis based on these data provide evidence for a ribbon α-conotoxin binding mode that might be exploited to design ligands with optimized selectivity.

Purification and characterization of thermostable l-asparaginase from Bacillus amyloliquefaciens MKSE in Korean soybean paste

This study aimed to produce and characterize thermostable l-asparaginase from Bacillus amyloliquefaciens MKSE, a newly screened strain from Korean fermented soybean paste. The isolated MKSE was identified by its morphological, biochemical, and physiological properties and 16S rRNA sequence. Its l-asparaginase II-like gene was expressed in Escherichia coli BL21 (DE3), by using the pET-28a (+) vector. The produced l-asparaginase (specific activity of 4.75 IU/mg) was purified by a one-step procedure, using nickel-affinity chromatography, and its molecular weight of 38.2 kDa was determined by gel electrophoresis. Its optimum pH and temperature were 8.5 and 65 °C, respectively. The enzyme was highly stable in the pH range of 3.0–11.0 for 6 h incubation, and the enzyme retained about 57% of its activity at 95 °C for 6 h. Its activity was activated in the presence of Mn2+, Mg2+, and Fe2+ while it was inhibited by Zn2+, Cu2+, PMSF, and SDS. Its Km was 1.5 mM, and its Vmax was 136.3 IU/mg. The results of this study reveal that MKSE l-asparaginase has a great potential to be used in various industries, due to its stability at high temperature and over a wide pH range.

Expression and purification of Class D flavin dependent monooxygenase N‐oxygenases

Members of the Class D flavin monooxygenase family are found in numerous natural product biosynthetic pathways, including those of valanimycin and daunorubicin, two medicinally useful natural products. Our lab is interested in understanding catalysis by this enzyme family. We are investigating several representative Class D monooxygenases, with the specific goal of understanding structure‐function relationships in their active sites. Our ultimate goal is to enable rational design of the enzymes in this family. The research presented here summarizes our efforts towards expression, purification and initial biochemical characterization of nitrososynthase from Streptomyces peucetius (DnmZ), and two isobutylamine N‐hydroxylase homologs (from Streptomyces setae, vlmH6, and Streptomyces viridochromogenes, vlmH7). These three Class D monooxygenases catalyze a biosynthetic step common to multiple natural products – flavin‐dependent hydroxylation of a primary amine. Streptomyces setae vlmH was successfully overexpressed in E. coli cells. The highest levels of protein were obtained when cultures were induced with 0.5 mM IPTG and incubated at 18°C. DnmZ is likewise expressed in E. coli with an optimal IPTG concentration and induction temperature of 0.1mM and 25°C, respectively. Both proteins will be purified by nickel affinity chromatography in preparation for preliminary characterization.Support or Funding InformationThis research was made possible by funding through the National Institutes of Health (NIH) grant #1SC3GM122652.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization and Structure Determination of the Perilipin 5 N‐Terminal Domain

Many countries, including the United States, have been afflicted with a group of lipid‐related diseases including type II diabetes, obesity and non‐alcoholic fatty liver disease. The commonality behind these disorders is lipid storage and lipid storage is mediated in part by the perilipin proteins. The perilipin family are a group of five conserved genes that differ with respect to size of the proteins they encode, tissue expression and transcriptional regulation. The only member of the perilipin family to have significant protein structural data collected is perilipin 3, and this is only on the carboxyl terminus. The C‐terminus of perilipin 5 has a high sequence homology to that of perilipin 3. Less is known about the amino terminus. The central focus of this study is to characterize and determine the structure of the N‐terminal domain of perilipin 5. The amino terminal 104 amino acids of perilipin 5 were expressed with a 6‐His tag in E. coli. Optimization of the expression of the protein fragment was ascertained using an immuno‐dot blot that contained cell samples treated with various ITPG concentrations and temperatures. Isolation and purification of the protein will then be performed using nickel affinity chromatography for further structural analysis. Secondary and tertiary structures of the N‐terminal fragment were predicted using Phyre2 and RaptorX. These computer models predicted a predominantly alpha helical structure with the presence of two 4‐helix bundles. Overall, determination of the N‐terminal structure for the perilipin 5 protein will increase our understanding of the perilipin family and their functions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression, purification, and characterization of asparaginase II from Saccharomyces cerevisiae in Escherichia coli

l-asparaginase catalyzes the conversion of l-asparagine to l-aspartate and ammonium. This protein is an important therapeutic enzyme used for the treatment of acute lymphoblastic leukemia. In this study, the asparaginase II-encoding gene ASP3 from Saccharomyces cerevisiae was cloned into the expression vector pET28a in-fusion with a 6x histidine tag and was expressed in Escherichia coli BL21 (DE3) cells. The protein was expressed at a high level (225.6 IU/g cells) as an intracellular and soluble molecule and was purified from the supernatant by nickel affinity chromatography. The enzyme showed very low activity against l-glutamine. The denaturing electrophoresis analysis indicated that the recombinant protein had a molecular mass of ∼38 kDa. The native enzyme was a tetramer with a molecular mass of approximately 178 kDa. The enzyme preparation showed antitumor activity against the K562 and Jurkat cell lines comparable or even superior to the E. coli commercial asparaginase.

Expression, purification and virucidal activity of two recombinant isoforms of phospholipase A2 from Crotalus durissus terrificus venom.

The global emergence and re-emergence of arthropod-borne viruses (arboviruses) over the past four decades have become a public health crisis of international concern, especially in tropical and subtropical countries. A limited number of vaccines against arboviruses are available for use in humans; therefore, there is an urgent need to develop antiviral compounds. Snake venoms are rich sources of bioactive compounds with potential for antiviral prospection. The major component of Crotalus durissus terrificus venom is a heterodimeric complex called crotoxin, which is constituted by an inactive peptide (crotapotin) and a phospholipase A2 (PLA2-CB). We showed previously the antiviral effect of PLA2-CB against dengue virus, yellow fever virus and other enveloped viruses. The aims of this study were to express two PLA2-CB isoforms in a prokaryotic system and to evaluate their virucidal effects. The sequences encoding the PLA2-CB isoforms were optimized and cloned into a plasmid vector (pG21a) for recombinant protein expression. The recombinant proteins were expressed in the E. coli BL21(DE3) strain as insoluble inclusion bodies; therefore, the purification was performed under denaturing conditions, using urea for protein solubilization. The solubilized proteins were applied to a nickel affinity chromatography matrix for binding. The immobilized recombinant proteins were subjected to an innovative protein refolding step, which consisted of the application of a decreasing linear gradient of urea and dithiothreitol (DTT) concentrations in combination with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) as a protein stabilizer. The refolded recombinant proteins showed phospholipase activity and virucidal effects against chikungunya virus, dengue virus, yellow fever virus and Zika virus.