Soluble overexpression and purification of bioactive human CCL2 in E. coli by maltose-binding protein.

Human chemokine (C-C motif) ligand 2 (hCCL2) is a small cytokine in the CC chemokine family that attracts monocytes, memory T lymphocytes, and natural killer cells to the site of tissue injury- or infection-induced inflammation. hCCL2 has been implicated in the pathogeneses of diseases characterized by monocytic infiltrates, including psoriasis, rheumatoid arthritis, atherosclerosis, multiple sclerosis, and insulin-resistant diabetes. The prokaryotic overexpression of hCCL2 has been investigated previously in an attempt to develop biomedical applications for this factor, but this has been hampered by protein misfolding and aggregation into inclusion bodies. In our present study, we screened 7 protein tags-Trx, GST, MBP, NusA, His8, PDI, and PDIb'a'-for their ability to allow the soluble overexpression of hCCL2. Three tags-MBP, His8, and PDI-solubilized more than half of the expressed hCCL2 fusion proteins. Lowering the expression temperature to 18 °C significantly further improved the solubility of all fusion proteins. MBP was chosen for further study based on its solubility, expression level, ease of purification, and tag size. MBP-CCL2 was purified using conventional chromatography and cleaved using TEV or Factor Xa proteases. Biological activity was assessed using luciferase and cell migration assays. Factor Xa-cleaved hCCL2 was found to be active and TEV-cleaved hCCL2 showed relatively less activity. This is probably because the additional glycine residues present at the N-terminus of hCCL2 following TEV digestion interfere with the binding of hCCL2 to its receptor.

Prokaryotic soluble overexpression and purification of bioactive human growth hormone by fusion to thioredoxin, maltose binding protein, and protein disulfide isomerase.

Human growth hormone (hGH) is synthesized by somatotroph cells of the anterior pituitary gland and induces cell proliferation and growth. This protein has been approved for the treatment of various conditions, including hGH deficiency, chronic renal failure, and Turner syndrome. Efficient production of hGH in Escherichia coli (E. coli) has proven difficult because the E. coli-expressed hormone tends to aggregate and form inclusion bodies, resulting in poor solubility. In this study, seven N-terminal fusion partners, hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A (NusA), protein disulfide bond isomerase (PDI), and the b'a' domain of PDI (PDIb'a'), were tested for soluble overexpression of codon-optimized hGH in E. coli. We found that MBP and hPDI tags significantly increased the solubility of the hormone. In addition, lowering the expression temperature to 18°C also dramatically increased the solubility of all the fusion proteins. We purified hGH from MBP-, PDIb'a'-, or Trx-tagged hGH expressed at 18°C in E. coli using simple chromatographic techniques and compared the final purity, yield, and activity of hGH to assess the impact of each partner protein. Purified hGH was highly pure on silver-stained gel and contained very low levels of endotoxin. On average, ∼37 mg, ∼12 mg, and ∼7 mg of hGH were obtained from 500 mL-cell cultures of Trx-hGH, MBP-hGH, and PDIb'a'-hGH, respectively. Subsequently, hGH was analyzed using mass spectroscopy to confirm the presence of two intra-molecular disulfide bonds. The bioactivity of purified hGHs was demonstrated using Nb2-11 cell.

Soluble expression and partial purification of recombinant human erythropoietin from E. coli.

Human erythropoietin (hEpo) is an essential regulator of erythrocyte production that induces the division and differentiation of erythroid progenitor cells in the bone marrow into mature erythrocytes. It is widely used for the treatment of anemia resulting from chronic kidney disease, chemotherapy, and cancer-related therapies. Active hEpo, and hEpo analogs, have been purified primarily from mammalian cells, which has several disadvantages, including low yields and high production costs. Although an Escherichia coli (E. coli) expression system may provide economic production of therapeutic proteins, it has not been used for the production of recombinant hEpo (rhEpo) because it aggregates in inclusion bodies in the E. coli cytoplasm and is not modified post-translationally. We investigated the soluble overexpression of active rhEpo with various protein tags in E. coli, and found out that several tags increased the solubility of rhEpo. Among them maltose binding protein (MBP)-tagged rhEpo was purified using affinity and gel filtration columns. Non-denaturing electrophoresis and MALDI-TOF MS analysis demonstrated that the purified rhEpo had two intra-disulfide bonds identical to those of the native hEpo. An in vitro proliferation assay showed that rhEpo purified from E. coli had similar biological activity as rhEpo derived from CHO cells. Therefore, we report for the first time that active rhEpo was overexpressed as a soluble form in the cytoplasm of E. coli and purified it in simple purification steps. We hope that our results offer opportunities for progress in rhEpo therapeutics.

Soluble expression of human leukemia inhibitory factor with protein disulfide isomerase in Escherichia coli and its simple purification.

Human leukemia inhibitory factor (hLIF) is a multifunctional cytokine that is essential for maintaining the pluripotency of embryonic stem cells. hLIF may be also be useful in aiding fertility through its effects on increasing the implantation rate of fertilized eggs. Thus these applications in biomedical research and clinical medicine create a high demand for bioactive hLIF. However, production of active hLIF is problematic since eukaryotic cells demonstrate limited expression and prokaryotic cells produce insoluble protein. Here, we have adopted a hybrid protein disulfide isomerase design to increase the solubility of hLIF in Escherichia coli. Low temperature expression of hLIF fused to the b'a' domain of protein disulfide isomerase (PDIb'a') increased the soluble expression in comparison to controls. A simple purification protocol for bioactive hLIF was established that includes removal of the PDIb'a' domain by cleavage by TEV protease. The resulting hLIF, which contains one extra glycine residue at the N-terminus, was highly pure and demonstrated endotoxin levels below 0.05 EU/µg. The presence of an intramolecular disulfide bond was identified using mass spectroscopy. This purified hLIF effectively maintained the pluripotency of a murine embryonic stem cell line. Thus we have developed an effective method to produce a pure bioactive version of hLIF in E. coli for use in biomedical research.

Expression and purification of biologically active human FGF2 containing the b'a' domains of human PDI in Escherichia coli.

Among the members of the fibroblast growth factor (FGF) family that affect the growth, differentiation, migration, and survival of many cell types, FGF2 is the most abundant in the central nervous system. Because of its wound healing effects, FGF2 has potential as a therapeutic agent. The protein is also added to the culture media to maintain stem cells. Expression and purification procedures for FGF2 that are highly efficient and low cost have been intensively investigated for the past two decades. Our current study focuses on the purification of FGF2 fused with b'a' domains of human protein disulfide isomerase to elevate overexpression, solubility, and stability with a simplified experimental procedure using only ion exchange chromatography, as well as on the confirmation of the biological activity of FGF2 on fibroblast Balb/c 3T3 cells and hippocampal neural cells.

Structural basis of cooperativity in human UDP-glucose dehydrogenase.

BACKGROUND: UDP-glucose dehydrogenase (UGDH) is the sole enzyme that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid. The product is used in xenobiotic glucuronidation in hepatocytes and in the production of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers, while inhibition of UGDH diminished tumor angiogenesis in vivo. A better understanding of the conformational changes occurring during the UGDH reaction cycle will pave the way for inhibitor design and potential cancer therapeutics. METHODOLOGY: Previously, the substrate-bound of UGDH was determined to be a symmetrical hexamer and this regular symmetry is disrupted on binding the inhibitor, UDP-α-D-xylose. Here, we have solved an alternate crystal structure of human UGDH (hUGDH) in complex with UDP-glucose at 2.8 Å resolution. Surprisingly, the quaternary structure of this substrate-bound protein complex consists of the open homohexamer that was previously observed for inhibitor-bound hUGDH, indicating that this conformation is relevant for deciphering elements of the normal reaction cycle. CONCLUSION: In all subunits of the present open structure, Thr131 has translocated into the active site occupying the volume vacated by the absent active water and partially disordered NAD+ molecule. This conformation suggests a mechanism by which the enzyme may exchange NADH for NAD+ and repolarize the catalytic water bound to Asp280 while protecting the reaction intermediates. The structure also indicates how the subunits may communicate with each other through two reaction state sensors in this highly cooperative enzyme.

Expression, high cell density culture and purification of recombinant EC-SOD in Escherichia coli.

Superoxide dismutase (SOD) catalyzes the dismutation of the biologically toxic superoxide anion into oxygen and hydrogen peroxide and is deployed by the immune system to kill invading microorganisms. Extracellular SOD (EC-SOD) is a copper- and zinc-containing glycoprotein found predominantly in the soluble extracellular compartment that consists of approximately 30-kDa subunits. Here, we purified recombinant EC-SOD3 (rEC-SOD) from Escherichia coli BL21(DE3) expressing a pET-SOD3-1 construct. Cells were cultured by high-density fed-batch fermentation to a final OD(600) of 51.8, yielding a final dry cell weight of 17.6 g/L. rEC-SOD, which was expressed as an inclusion body, comprised 48.7% of total protein. rEC-SOD was refolded by a simple dilution refolding method and purified by cation-exchange and reverse-phase chromatography. The highly purified rEC-SOD thus obtained was a mixture of monomers and dimers, both of which were active. The molecular weights of monomeric and dimeric rEC-SOD were 25,255 and 50,514 Da, respectively. The purified rEC-SOD had 4.3 EU/mg of endotoxin and the solubility of rEC-SOD was more than 80% between pH 7 and 10. In 2 L of fed-batch fermentation, 60 mg of EC-SOD (99.9% purity) could be produced and total activity was 330.24 U. The process established in this report, involving high-cell-density fermentation, simple dilution refolding, and purification with ion-exchange and reverse-phase chromatography, represents a commercially viable process for producing rEC-SOD.

H(1) antihistamine drug promethazine directly blocks hERG K(+) channel.

Promethazine is a phenothiazine derivative with antihistaminic (H(1)), sedative, antiemetic, anticholinergic, and antimotion sickness properties that can induce QT prolongation, which may lead to torsades de pointes. Since block of cardiac human ether-a-go-go-related gene (hERG) channels is one of the leading causes of acquired long QT syndrome, we investigated the acute effects of promethazine on hERG channels to determine the electrophysiological basis for its proarrhythmic potential. Promethazine increased the action potential duration at 90% of repolarization (APD(90)) in a concentration-dependent manner, with an IC(50) of 0.73microM when action potentials were elicited under current clamp in guinea pig ventricular myocytes. We examined the effects of promethazine on the hERG channels expressed in Xenopus oocytes and HEK293 cells using two-microelectrode voltage-clamp and patch-clamp techniques. Promethazine induced a concentration-dependent decrease of the current amplitude at the end of the voltage steps and hERG tail currents. The IC(50) of promethazine dependent hERG block in Xenopus oocytes decreased progressively relative to the degree of depolarization. The IC(50) for the promethazine-induced block of the hERG currents in HEK293 cells at 36 degrees C was 1.46microM at +20mV. Promethazine affected the channels in the activated and inactivated states but not in the closed states. The S6 domain mutations, Y652A and F656A partially attenuated (Y652A) or abolished (F656A) the hERG current block. These results suggest that promethazine is a blocker of the hERG channels, providing a molecular mechanism for the arrhythmogenic side effects during the clinical administration of promethazine.

Computational analysis of the quaternary structural changes induced by point mutations in human UDP-glucose dehydrogenase.

UDP-glucose dehydrogenase (UGDH) is an enzyme catalyzing the conversion of UDP-glucose to UDP-glucuronic acid. Site-directed mutagenesis studies have revealed that human UGDH (hUGDH) has distinct oligomeric states that vary with different point mutations. In this study we have investigated how the changes in the oligomer-forming propensity may be involved in the thermal motion of wild-type hUGDH and its mutants, using normal mode analysis (NMA). Our results show that the perturbation caused by the mutation of a residue at a considerably distant location from the oligomeric interfaces is preferentially distributed throughout specific sites, especially the large flexible regions in the hUGDH structure, thereby changing the motional fluctuation pattern at the oligomeric interfaces. A large-magnitude cooperative motion at the oligomeric interfaces is a critical factor in interfering with the hexamer formation of the enzyme. In particular, structural stability at the dimeric interface is necessary to retain the hexameric structure of hUGDH.

Clomipramine block of the hERG K+ channel: accessibility to F656 and Y652.

Clomipramine is a tricyclic antidepressant for psychiatric disorders that can induce QT prolongation, which may lead to torsades de pointes. Since blockade of cardiac human ether-a-go-go-related gene (hERG) channels is an important cause of acquired long QT syndrome, we investigated the acute effects of clomipramine on hERG channels to determine the electrophysiological basis for its proarrhythmic potential. We examined the effects of clomipramine on the hERG channels expressed in Xenopus oocytes and HEK293 cells using two-microelectrode voltage-clamp and patch-clamp techniques. Clomipramine induced a concentration-dependent decrease in the current amplitude at the end of the voltage steps and hERG tail currents. The IC50 for clomipramine needed to block the hERG current in Xenopus oocytes decreased progressively relative to the degree of depolarization. The fractional electrical distance was estimated to be delta=0.83. The IC50 for the clomipramine-induced blockade of the hERG currents in HEK293 cells at 36 degrees C was 0.13 microM at +20 mV. Clomipramine affected the channels in the activated and inactivated states but not in the closed states. The clomipramine-induced blockade of hERG was found to be use-dependent, exhibiting a more rapid onset and a greater steady-state block at the higher frequencies of activation. The S6 domain mutations, Y652A and F656A partially attenuated (Y652A) or abolished (F656A) the hERG-current blockade. These results suggest that clomipramine is a blocker of the hERG channels, providing a molecular mechanism for the arrhythmogenic side effects during the clinical administration of clomipramine.

Protriptyline block of the human ether-à-go-go-related gene (HERG) K+ channel.

Protriptyline, a tricyclic antidepressant for psychiatric disorders, can induce prolonged QT, torsades de pointes, and sudden death. We studied the effects of protriptyline on human ether-à-go-go-related gene (HERG) channels expressed in Xenopus oocytes and HEK293 cells. Protriptyline induced a concentration-dependent decrease in current amplitudes at the end of the voltage steps and HERG tail currents. The IC(50) for protriptyline block of HERG current in Xenopus oocytes progressively decreased relative to the degree of depolarization, from 142.0 microM at -40 mV to 91.7 microM at 0 mV to 52.9 microM at +40 mV. The voltage dependence of the block could be fit with a monoexponential function, and the fractional electrical distance was estimated to be delta=0.93. The IC(50) for the protriptyline-induced blockade of HERG currents in HEK293 cells at 36 degrees C was 1.18 microM at +20 mV. Protriptyline affected channels in the activated and inactivated states, but not in the closed states. HERG blockade by protriptyline was use-dependent, exhibiting a more rapid onset and a greater steady-state block at higher frequencies of activation. Our findings suggest that inhibition of HERG currents may contribute to the arrhythmogenic side effects of protriptyline.

Maprotiline block of the human ether-a-go-go-related gene (HERG) K+ channel.

Maprotiline, an atypical antidepressant, can induce prolonged QT and torsades de pointes. We studied the effects of maprotiline on human ether-a-go-go-related gene (HERG) channels expressed in Xenopus oocytes and HEK293 cells. Maprotiline induced a concentration-dependent decrease in current amplitudes at the end of the voltage steps and tail currents of HERG. The V1/2 values in the absence and presence of 1-20 microM maprotiline were not significantly different, while the values decreased according to the concentrations of the drug at 50-300 microM. The IC50 for a maprotiline block of HERG current in Xenopus oocytes did not change according to depolarization; 39.5 +/- 3.2 microM at -40 mV and 43.6 +/- 2.8 microM at +40 mV. The block of HERG by maprotiline was examined after treatment of trinitrobenzene sulfonic acid (TNBS), an amino-group reagent that neutralizes the positively charged amino-groups of peptide N-terminal and lysine residues. TNBS inhibited the change of V1/2 values induced by 50-300 mM maprotiline, and aggravated the drug-induced gmax decrease. The IC50 for the maprotiline-induced blockade of HERG currents in HEK293 cells at 36 degrees C was 0.13 microM at +20 mV. Our findings suggest that the arrhythmogenic side effects of maprotiline are caused by a blockade of HERG and possibly by a blockade of delayed rectifier K+ channel.