Characterization of two human flavin-containing monooxygenase (form 3) enzymes expressed in Escherichia coli as maltose binding protein fusions

To examine the possibility for drug metabolism polymorphism, adult human flavin-containing monooxygenases (form 3) (EC 1.14.13.8) that differ at one amino acid were expressed in Escherichia coli as maltose binding protein fusions. The cDNA that was first reported during the cloning of adult human flavin-containing monooxygenase was designated the wild type lys158 enzyme. A second cDNA has been identified as a common polymorphism in some human populations and was designated the glu158 enzyme. The cDNA that encodes both enzymes was subcloned into a high yield protein fusion expression system, expressed, and the protein was partially purified by affinity chromatography and characterized for enzyme activity with selective functional substrate probes. N- and S-oxygenation activity of both enzymes was determined with 10-(N,N-dimethylaminopentyl)-2-(trifluoromethyl)phenothiazine and methyl p-tolyl sulfide, respectively. It was found that expression of both lys158 and glu158 enzymes of the human flavin-containing monooxygenase form 3 as fusions with the maltose binding protein resulted in an enzyme that was soluble and greatly stabilized and had a reduced requirement for detergent during enzyme purification and during the assay for activity. Expression of the fusion proteins has allowed the preparation of stable and highly active enzyme at greater purity than was readily possible in the past. With the exception of the stability and solubility characteristics, the physical and chemical properties of lys158 and glu158 maltose binding fusion proteins of human flavin-containing monooxygenase form 3 variants resembled that of flavin-containing monooxygenase enzyme activity associated with human liver microsomes and enzyme isolated from a previous Escherichia coli expression system that lacked the protein fusion. Comparison of the catalytic activity of the two fusion proteins showed that while both forms were active, there were differences in their substrate specificities. Expression of the adult human flavin-containing monooxygenase form 3 as a maltose binding protein has allowed considerable advances over the previously reported cDNA-expressed enzyme systems and may provide the basis for examining the role of the flavin-containing monooxygenase in human xenobiotic or drug metabolism.     

Purification and crystallization of the CDK-associated protein phosphatase KAP expressed in Escherichia coli

The kinase associated phosphatase (KAP) is a human dual specificity protein phosphatase that dephosphorylates the cell cycle control protein, cyclin dependent kinase-2 on Thr 160 in a cyclin dependent manner (Poon & Hunter, 1995). We report here the over-expression of KAP in Escherichia coli as an N-terminal His-tagged protein using a modified pET-28a T7-expression vector. The recombinant protein was purified to homogeneity and crystallized. The crystals diffract to 2.3 A resolution when exposed to synchrotron radiation and belong to space group P6(1)22, or its enantiomorph P6(5)22, with unit cell dimensions a = b = 74.5 A, c = 139.5 A.     

Purification and characterization of D-beta-hydroxybutyrate dehydrogenase expressed in Escherichia coli

D-beta-Hydroxybutyrate dehydrogenase (BDH), a lipid-requiring enzyme, has been cloned into pUC18, expressed in Escherichia coli, and purified to homogeneity. The apoenzyme, i.e., the enzyme devoid of phospholipid, has no activity, but can be activated by phospholipid to a specific activity of 129 mumol/(min.mg). The functional properties of the enzyme expressed in E. coli were compared with the enzyme purified from rat liver. The specific activities, kinetic parameters, and phospholipid activation profiles were virtually identical. These results indicate that the expression of the enzyme in E. coli is a viable method for producing active functional BDH and should allow for the production of specifically altered BDH molecules.     

One-step purification of plant ferredoxin-NADP+ oxidoreductase expressed in Escherichia coli as fusion with glutathione S-transferase

Complementary DNA sequences encoding the mature form of pea ferredoxin-NADP+ reductase were cloned in-frame at the 3' end of the Schistosoma japonicum glutathione S-transferase gene in the expression vector pGEX-3X (Smith and Johnson, Gene 67, 31-40, 1988). A spacer sequence linking the two genes was modified to provide a proteolytic site just before the first amino acid residue of mature pea reductase. When introduced into competent Escherichia coli cells and induced, the resulting plasmid (pGF205) directed the expression of a 60-kDa immunoreactive peptide that results from the fusion between glutathione S-transferase and ferredoxin-NADP+ reductase sequences. The fused protein could be purified in a single step by selective absorption onto glutathione-agarose beads, followed by elution with free glutathione. It showed both transferase and reductase activities. Removal of the transferase portion by cleavage with the restriction protease Xa rendered ferredoxin-NADP+ reductase electrophoretically homogeneous. The purified transgenic enzyme showed kinetic and spectroscopic properties that were similar to those reported for the plant flavoprotein, indicating that, even when fused to the 27-kDa transferase portion, the reductase was still able to assemble FAD and to acquire an active conformation in the bacterial host. The expression-purification protocol employed here allows the isolation of up to 1 mg of active ferredoxin-NADP+ reductase/g of transformed cells. The system is potentially useful for the purification of activity-impaired forms of the flavoprotein.     

Mutation of a single MalK subunit severely impairs maltose transport activity in Escherichia coli

The maltose transport system of Escherichia coli, a member of the ABC transport superfamily of proteins, consists of a periplasmic maltose binding protein and a membrane-associated translocation complex that contains two copies of the ATP-binding protein MalK. To examine the need for two nucleotide-binding domains in this transport complex, one of the two MalK subunits was inactivated by site-directed mutagenesis. Complexes with mutations in a single subunit were obtained by attaching a polyhistidine tag to the mutagenized version of MalK and by coexpressing both wild-type MalK and mutant (His)6MalK in the same cell. Hybrid complexes containing one mutant (His)6MalK subunit and one wild-type MalK subunit were separated from those containing two mutant (His)6MalK proteins based on differential affinities for a metal chelate column. Purified transport complexes were reconstituted into proteoliposome vesicles and assayed for maltose transport and ATPase activities. When a conserved lysine residue at position 42 that is involved in ATP binding was replaced with asparagine in both MalK subunits, maltose transport and ATPase activities were reduced to 1% of those of the wild type. When the mutation was present in only one of the two subunits, the complex had 6% of the wild-type activities. Replacement of a conserved histidine residue at position 192 in MalK with arginine generated similar results. It is clear from these results that two functional MalK proteins are required for transport activity and that the two nucleotide-binding domains do not function independently to catalyze transport.     

Calcium and membrane binding properties of bovine neurocalcin delta expressed in Escherichia coli

Neurocalcins are brain-specific proteins that belong to a new subclass of the EF-hand superfamily of calcium binding proteins, defined by the photoreceptor cell-specific protein, recoverin. Recoverin, which regulates the desensitization of photo-excited rhodopsin, is myristoylated and exhibits a calcium-myristoyl switch. Like recoverin, neurocalcins have a signal for N-myristoylation and possess four EF-hands, although the first one lacks some residues critical for calcium binding. In this work, I have examined the calcium and membrane binding properties of recombinant myristoylated and unmyristoylated neurocalcin delta. I show that neurocalcin, like recoverin, binds to biological membranes in a calcium- and myristoyl-dependent manner. Both myristoylated and unmyristoylated proteins bind three calcium ions. However, the unmyristoylated form exhibits a higher affinity for calcium than the myristoylated protein but shows a lower cooperativity in binding calcium. These data support the model for the calcium-myristoyl switch mechanism proposed for recoverin (Zozulya, S., and Stryer, L. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11569-11573; Dizhoor, A. M., Chen, C. K., Olshevskaya, E., Sinelnikova, V. V., and Hurley, J. B. (1993) Science 259, 829-832). Using point mutations, I have investigated the relative importance of each of the three functional EF hands (EF2, EF3, and EF4) in the calcium and membrane binding properties of neurocalcin. Calcium and membrane binding properties of the mutant-myristoylated proteins suggest that binding of calcium to EF2 is critical in triggering the binding of the protein to membranes.     

Purification of recombinant human rhinovirus 14 3C protease expressed in Escherichia coli

A physiologically relevant thrombopoietin (TPO) must be a humoral regulator with lineage specificity for megakaryocytes and their precursors. It should be capable of stimulating platelet production in normal animals, and elevated levels of TPO should be detectable in the plasma following acute, severe thrombocytopenia. Acute thrombocytopenia provides a model system that is likely to predict the effects of TPO, since many of the effects on megakaryocytes and platelets observed after induction of acute thrombocytopenia would be mediated by TPO. Important questions remain to be answered. Do the currently available data for the c-Mpl ligand explain previously published data that describe elevated levels of Meg-CSF in the circulation following production of bone marrow aplasia? Does the c-Mpl ligand account for all of the megakaryocyte stimulatory factors that have been described? Is there another factor that accounts for at least some of the acute alterations in megakaryocytopoiesis that occur immediately following a decrease in platelet levels?     

Purification and characterization of Streptococcus mutans glucosyltransferase (GtfC) expressed in Escherichia coli

Streptococcus mutans constitutively expresses three glucosyltransferases, i.e., GtfB, GtfC, and GtfD; which synthesize glucan polymers from sucrose. To obtain individual GTF without complexing with one another, a purification strategy was developed to recover recombinant GTF expressed from Escherichia coli. The recombinant GtfC was aggregated and associated with the insoluble fraction in E. coli homogenates. GtfC was solublized with the 8M urea, renatured to its biologically active form by serial dialysis against sodium phosphate buffer, and subsequently purified to homogeneity by DEAE-Sephacel and hydroxylapatite column chromatography. The GtfC enzyme preparation was purified 16.3-fold and the molecular weight was estimated to be 140 kDa. GtfC synthesized water insoluble glucan in a primer independent manner and its enzymatic activities could be enhanced by dextran. Purified GtfC had a pH optimum of 6.5, a K(m) of 9.26 mM for sucrose and a pI of 5.5. Distinct from the previous reports, results from this study offers an alternative for the purification of the recombinant GTFs free from any detergent contamination to make it more suitable for utilization in vivo.     

Diagnostic potential of Toscana virus N protein expressed in Escherichia coli

The nucleocapsid (N) protein of the Toscana (TOS) virus was expressed in Escherichia coli by using a pET15b vector. The recombinant protein was purified by affinity chromatography and was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and enzyme immunoassay (EIA). The recombinant antigen was reactive with positive human sera, and the reactivity correlated very well (r = 0.9) with that of a whole-virus antigen when tested by EIA with 30 TOS virus-positive and 30 TOS virus-negative serum samples. The results demonstrate that the recombinant N protein can be easily produced in a procaryotic system and used for diagnostic assays for TOS virus immunity.     

Purification and characterization of herpes simplex virus type 1 alkaline exonuclease expressed in Escherichia coli

The alkaline exonuclease (AE) encoded by the herpes simplex virus type 1 (HSV-1) UL12 open reading frame was inducibly expressed in Escherichia coli and purified without the use of chromatographic separation. This recombinant AE was found to exhibit the same biochemical properties as the virus-encoded protein and was used to confirm the existence of a weak endonucleolytic activity in the enzyme. Antisera raised against the recombinant protein recognized several forms of the AE in HSV-1-infected cells. This expression and purification strategy will provide an economical and easily accessible alternative source of HSV-1 AE for future in vitro studies.     

Ndd, the bacteriophage T4 protein that disrupts the Escherichia coli nucleoid, has a DNA binding activity

Early in a bacteriophage T4 infection, the phage ndd gene causes the rapid destruction of the structure of the Escherichia coli nucleoid. Even at very low levels, the Ndd protein is extremely toxic to cells. In uninfected E. coli, overexpression of the cloned ndd gene induces disruption of the nucleoid that is indistinguishable from that observed after T4 infection. A preliminary characterization of this protein indicates that it has a double-stranded DNA binding activity with a preference for bacterial DNA rather than phage T4 DNA. The targets of Ndd action may be the chromosomal sequences that determine the structure of the nucleoid.     

DNA binding and helicase domains of the Escherichia coli recombination protein RecG

The Escherichia coli RecG protein is a unique junction-specific helicase involved in DNA repair and recombination. The C-terminus of RecG contains motifs conserved throughout a wide range of DNA and RNA helicases and it is thought that this C-terminal half of RecG contains the helicase active site. However, the regions of RecG which confer junction DNA specificity are unknown. To begin to assign structure-function relationships within RecG, a series of N- and C-terminal deletions have been engineered into the protein, together with an N-terminal histidine tag fusion peptide for purification purposes. Junction DNA binding, unwinding and ATP hydrolysis were disrupted by mutagenesis of the N-terminus. In contrast, C-terminal deletions moderately reduced junction DNA binding but almost abolished unwinding. These data suggest that the C-terminus does contain the helicase active site whereas the N-terminus confers junction DNA specificity.     

Characterization of Escherichia coli NrdH. A glutaredoxin-like protein with a thioredoxin-like activity profile

Ribonucleotides are converted to deoxyribonucleotides by ribonucleotide reductases. Either thioredoxin or glutaredoxin is a required electron donor for class I and II enzymes. Glutaredoxins are reduced by glutathione, thioredoxins by thioredoxin reductase. Recently, a glutaredoxin-like protein, NrdH, was isolated as the functional electron donor for a NrdEF ribonucleotide reductase, a class Ib enzyme, from Lactococcus lactis. The absence of glutathione in this bacterium raised the question of the identity of the intracellular reductant for NrdH. Homologues of NrdH are present in the genomes of Escherichia coli and Salmonella typhimurium, upstream of the genes for the poorly transcribed nrdEF, separated from it by an open reading frame (nrdI) coding for a protein of unknown function. Overexpression of E. coli NrdH protein shows that it is a functional hydrogen donor with higher specificity for the class Ib (NrdEF) than for the class Ia (NrdAB) ribonucleotide reductase. Furthermore, this glutaredoxin-like enzyme is reduced by thioredoxin reductase and not by glutathione. We suggest that several uncharacterized glutaredoxin-like proteins present in the genomes of organisms lacking GSH, including archae, will also react with thioredoxin reductase and be related to the ancestors from which the GSH-dependent glutaredoxins have evolved by the acquisition of a GSH-binding site. We also show that NrdI, encoded by all nrdEF operons, has a stimulatory effect on ribonucleotide reduction.     

A nitrite biosensor based on a maltose binding protein nitrite reductase fusion immobilized on an electropolymerized film of a pyrrole-derived bipyridinium

The preparation and electrochemical characterization of glassy carbon electrodes (GCEs) modified with electropolymerized films of the cation N-(3-pyrrol-1-yl-propyl)-4,4'-bipyridine (PPB) are described. The behavior of a new biosensor, which exhibits a high catalytic activity for nitrite reduction and which consists of a maltose binding protein nitrite reductase fusion (MBP-Nir) immobilized on an electropolymerized film of PPB as an electrocatalyst, is also described. The insoluble perchlorate salt of the poly(benzyl viologen) dication was used to immobilize MBP-Nir onto an electrode previously modified with an electropolymerized film of PPB. The electropolymerized film of PPB on the GCE is redox active and exhibits special electron-transfer properties toward the MBP-Nir layer but not toward Nir (Nir without MBP fusion attached), suggesting an intimate interaction between the PPB film and the MBP-Nir layer. The kinetics of the catalytic reaction between the biosensor and nitrite anion were characterized using cyclic voltammetry and rotated disk electrode techniques, and a value of (4.6 +/- 0.5) x 10(3) M-1 S-1 was obtained for the rate constant.     

Functional domains of Escherichia coli single-stranded DNA binding protein as assessed by analyses of the deletion mutants

A series of C- and N-terminal deletion mutants of Escherichia coli single-stranded DNA binding protein (SSB) was constructed, purified, and characterized in terms of ability to self-multimerize and to bind to DNA. High-performance gel filtration chromatography revealed that the amino acids 89-105 play a key role in the maintenance of homotetramer for native SSB of 177 amino acids. Interestingly, all of the N-terminal deletion mutants studied here were eluted as octamers, indicating that the N-terminal 11 residues are involved in the prevention of the formation of octamers. The binding of SSB and its deletion mutant proteins to single-stranded d(T)n was examined by gel mobility shift assay and circular dichroism spectroscopy. C-terminal deletion mutant proteins, SSB1-135 and SSB1-115, maintained high affinity and may be wrapped by single-stranded DNA (ssDNA) in the same way as in the case of native SSB. In contrast, deletion of the C-terminal region (residues 89-115) or N-terminal region (residues 1-11) caused a dramatic decrease in the binding affinity. Furthermore, two different stoichiometries of SSB in the complexes with d(T)64, but not with d(T)32, were observed for native SSB, SSB1-135, SSB1-115, and SSB37-177, suggesting that the (SSB)65 and (SSB)35 binding modes, as previously demonstrated [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603; Bujalowski, W., & Lohman, T. M. (1986) Biochemistry 25, 7799-7802], occurred at lower and higher SSB concentrations, respectively. A functional map for SSB molecule was presented and discussed.     

Fungal beta-tubulin, expressed as a fusion protein, binds benzimidazole and phenylcarbamate fungicides

Benzimidazoles are important antitubulin agents used in veterinary medicine and plant disease control. Resistance is a practical problem correlated with single amino acid changes in beta-tubulin and is often linked to greater sensitivity to phenylcarbamates. This negative cross-resistance creates opportunities for durable antiresistance strategies. Attempts to understand the molecular basis of benzimidazole resistance have been hampered by the inability to purify tubulin from filamentous fungi. We have overcome some of these problems by expressing beta-tubulin as a fusion with a maltose binding protein. This fusion protein is soluble, and we confirm for the first time using a gel filtration assay that benzimidazoles indeed bind to beta-tubulin. This binding is reduced by the mutation Glu198-->Gly198, which also confers resistance. Binding of phenylcarbamates is the complete opposite, reflecting their biological activity and the negative cross-resistance. This suggests that the fungicide binding sites fold correctly in the fusion protein.     

Purification of the cardiac sarcoplasmic reticulum membrane protein phospholamban from recombinant Escherichia coli

Phospholamban (PLN) was expressed in Escherichia coli as a protein fusion with glutathione S-transferase (GST). GST-PLN was mostly present in the insoluble protein fraction and accounted for approximately 50% of total insoluble protein. Attempts to suppress inclusion body formation or to use GST as an affinity-purification tag failed. A successful purification method is based on preparative SDS/PAGE and electrodialysis. From 1 g cells we typically purified 13.5 mg fusion protein with a PLN content of 2.8 mg. We genetically inserted an enterokinase (EK) protease site just in front of the PLN sequence and demonstrated the proteolytical liberation of PLN from the carrier protein. The approach described represents a substantial advancement in PLN expression and purification.