Mass spectrometric characterization of the human androgen receptor ligand-binding domain expressed in Escherichia coli

The ligand-binding domain (LBD) of the human androgen receptor (hAR LBD), encompassing amino acids (AAs) 647-919, was expressed in Escherichia coli with an N-terminal polyhistidine tag (His(10)-hAR LBD) from a pET-16b vector. The overexpressed protein was initially insoluble in inclusion bodies, and was subsequently solubilized in 8 M guanidine hydrochloride (GdnHCl). The solubilized His(10)-hAR LBD was purified to apparent homogeneity by metal ion affinity chromatography in the presence of 6 M GdnHCl. The isolated protein migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular mass of 33-34 kDa, as expected from the plasmid construct. Immunoblot analysis with C-terminal antibodies raised against a peptide corresponding to the last 19 AAs (AAs 901-919) of hAR revealed that the purified protein contained an immunoreactive epitope present within the AR and was of the appropriate size. Further characterization, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), showed a single protein species of average mass 34 580 Da, confirming the size and purity of the purified His(10)-hAR LBD. Detailed tryptic peptide mapping analysis, using MALDI/TOF-MS, identified a total of eight peptides with a 30% coverage of the LBD, including the last tryptic peptide in the hAR sequence. These data confirm that the purified protein was the intact hAR LBD. AA sequencing of these tryptic peptides, using an HPLC-coupled electrospray ionization ion trap mass spectrometer (LC/ESI-ITMS and MS/MS), unambiguously confirmed that the peptides were from the hAR LBD. The purified His(10)-hAR LBD in 6 M GdnHCl could be renatured as determined by ligand-binding activity, with a similar equilibrium dissociation constant (K(d)) for [(3)H]-mibolerone and a similar steroid specificity to the AR isolated from rat ventral prostate.     

Expression of androgen receptor in insect cells. Purification of the receptor and renaturation of its steroid- and DNA-binding functions.

A full-length rat androgen receptor cDNA was used to produce a recombinant baculovirus (AcrAR) by homologous recombination. Spodoptera frugiperda (Sf9) cells infected with this virus expressed a 110-kDa polypeptide that amounted up to about one-third of total cell protein. Studies with AR antibodies confirmed that this protein was indeed rAR. Only a minor portion of the recombinant AR was soluble in buffers without ionic detergents, but its complete solubilization was achieved in 6 M guanidine HCl (GdnHCl). Electron microscopy of cell pellets revealed that AR was localized to electron-dense cytoplasmic aggregates. The soluble cytosolic receptor was biologically active, in that it bound [3H]mibolerone with high affinity and specificity and interacted with an androgen-responsive element. The functions of the GdnHCl-solubilized AR were partially restored by a 20-50-fold dilution. The solubilized receptor was purified to an apparent homogeneity in a single step by gel filtration on a Sephacryl S-400 column in the presence of 6 M GdnHCl. The homogeneous AR protein could be renatured to bind [3H]mibolerone, interact specifically with a DNA element, and be recognized by receptor antibodies. Receptor-DNA interaction was stabilized by an antibody directed against the N-terminal part and abolished by an antibody against the hinge region of the receptor Zn2+ ions were essential for the purified receptor to refold into a specific DNA-binding form during the renaturation, with the optimal ZnCl2 concentration being 50-100 microM depending on the buffer conditions. Cd2+ ions were also capable of restoring the receptor's DNA-binding activity and did so at concentrations 10-fold lower than those of the Zn2+ ions.     

Identification of a novel phosphorylation site in human androgen receptor by mass spectrometry

An N-terminal hexahistidine-tagged full-length human androgen receptor protein (His(6)-hAR) was overexpressed and purified to apparent homogeneity in the presence of dihydrotestosterone (DHT) in our previous studies. In-gel trypsin digestion of the purified DHT-bound His(6)-hAR, and tryptic peptide mapping using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), detected a total of 17 peptides (21% coverage of hAR) with 9 peptides originating from the ligand-binding domain (LBD, 31% coverage of LBD). Amino acid sequencing analysis of the tryptic peptides from a separate in-gel digestion of the His(6)-hAR, using HPLC-coupled electrospray ionization ion trap mass spectrometry (LC/ESI-ITMS and MS/MS), unambiguously confirmed 21 peptides with 19% coverage of the hAR, of which 11 peptides originated from the LBD (35% coverage of LBD). These 21 peptides included 11 out of the 17 peptides detected by MALDI/TOF-MS. In addition, a novel serine phosphorylation site (Ser(308)) within the N-terminal transactivation domain of hAR was identified.     

Purification and characterization of an epoxide hydrolase from the peroxisomal fraction of mouse liver

Epoxide hydrolase (EH) activity has been reported to occur in most subcellular fractions of mouse liver. The EHs in the microsomal and cytosolic fractions have been purified and characterized; however, the nature of the EH(s) in the peroxisomal fraction is not known. Therefore an EH, pEH, was purified from the solubilized 12,000g fraction, which contain peroxisomes. Previous studies have demonstrated that the EH activity in this crude solubilized 12,000g fraction resides mostly in the peroxisomes. Thus the crude 12,000g pellet from mouse liver, free from cytosolic contamination, was sonicated to obtain a 105,000g soluble fraction containing 80% of the original EH activity in this fraction. The pEH was purified, using trans-stilbene oxide (TSO) as substrate, by a combination of affinity and hydroxyapatite chromatography. The purified pEH had a native molecular weight of 57 kDa, a molecular weight of 59 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 5.7. The purified pEH was observed to be immunologically similar to the cytosolic EH (cEH). The kinetics of hydrolysis of TSO, however, were slightly different. Lineweaver-Burk plots for the inhibition of pEH suggest a probable noncompetitive, mixed-type inhibition. The purified pEH thus appears to be very similar to the cEH. There are minor differences between the purified cEH and pEH, particularly in the kinetic parameters. However, these minor differences are insignificant. These results demonstrate that the cEH and pEH are substantially similar, if not identical.     

Purification of insoluble nitric oxide synthase from rat cerebellum.

Nitric oxide synthase [EC 1.14.23] from the particulate fraction of rat cerebella was purified and characterized. The homogenate of rat cerebella was centrifuged to obtain a pellet, which was washed and incubated with Triton X-100 containing buffer. The enzyme activity appeared in the 100,000 x g supernatant after incubation with the detergent. The solubilized enzyme was then purified by sequential affinity chromatography using adenosine 2',5'-diphosphate agarose and calmodulin Sepharose 4B, which gave a product that migrated as a single protein band on SDS/PAGE with a molecular mass of about 150 kDa. The purified enzyme exhibited an absolute requirement for FAD, in addition to NADPH and Ca2+/calmodulin. Thus, there is an insoluble nitric oxide synthase in rat cerebellum that has similar characteristics to the soluble type.     

Characterization of multiple forms of folate-binding protein from human leukemia cells

Folate-binding proteins were isolated from the particulate fraction (44,000 X g pellet) and the soluble fraction (44,000 X g supernate) of the homogenate of a spleen obtained from a patient who had an acute leukemic (blast) transformation of chronic myelogenous leukemia. The folate-binding activity which was obtained from the particulate fraction by solubilization with 1% Triton X-100 could be resolved into two binding proteins (Mr 310,000 and 28,000) by gel filtration through Sephadex G-200 after incubation with excess [3H]pteroylglutamic acid (PteGlu). The folate-binding protein in the solubilized particulate fraction and the soluble folate-binding protein in the 44,000 X g supernatant cytoplasm were purified by affinity chromatography. Only a 32 kDa protein was identified by SDS-polyacrylamide gel electrophoresis in the final preparation of the purified folate-binding protein from the particulate, whereas two protein bands (Mr 42,000 and 32,000) were identified by SDS-polyacrylamide gel electrophoresis in the purified preparation of the soluble folate-binding protein. Both of these species were immunologically crossreacting. Both the purified folate-binding protein from the particulate fraction and the purified soluble form had higher affinity for oxidized folate than for the reduced folate cofactors, and both proteins had very low affinity for the antifolate compound, methotrexate. The amino-acid composition of the soluble folate-binding protein was similar with regard to the content of apolar amino acids to that reported for the membrane-derived folate-binding protein purified from milk and human placenta.     

Affinity chromatography of RecA protein and RecA nucleoprotein complexes on RecA protein-agarose columns

We have analyzed the nature of RecA protein-RecA protein interactions using an affinity column prepared by coupling RecA protein to an agarose support. When radiolabeled soluble proteins from Escherichia coli are applied to this column, only the labeled RecA protein from the extract was selectively retained and bound tightly to the affinity column. Efficient binding of purified 35S-labeled RecA protein required Mg2+, and high salt did not interfere with the binding of RecA protein to the column. Complete removal of the bound enzyme from the affinity column required treatment with guanidine HCl (5 M) or urea (8 M). These and other properties suggest that hydrophobic interactions contribute significantly to RecA protein subunit recognition in solution. Using a series of truncated RecA proteins synthesized in vitro, we have obtained evidence that at least some of the sequences involved in protein recognition are localized within the first 90 amino-terminal residues of the protein. Based on the observation that RecA proteins from three heterologous bacteria are specifically retained on the E. coli RecA affinity column, it is likely that this binding domain is highly conserved and is required for interaction and association of RecA protein monomers. Stable ternary complexes of RecA protein and single-stranded DNA were formed in the presence of the nonhydrolyzable ATP analog adenosine 5'-O-(thiotriphosphate) and applied to the affinity columns. Most of the complexes formed with M13 DNA could be eluted in high salt, whereas a substantial fraction of those formed with the oligonucleotide (dT)25-30 remained bound in high salt and were quantitatively eluted with guanidine HCl (5 M). The different binding properties of these RecA protein-DNA complexes likely reflect differences in the availability of a hydrophobic surface on RecA protein when it is bound to long polynucleotides compared to short oligonucleotides.     

Solubilization and partial purification of GABAB receptor from bovine brain

gamma-Aminobutyric acid (GABA)B receptor has been solubilized and partially purified by an affinity column chromatography. GABAB receptor was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in the presence of asolectin. The solubilized GABAB receptor was adsorbed on baclofen-coupled epoxy-activated Sepharose 6B. The affinity matrix adsorbed 80% of the solubilized [3H]GABA binding activity to GABAB receptor, and approximately 75% of the adsorbed activity could be eluted with 1 M KC1. GABAB receptor binding in the fraction eluted from affinity column was displaced by GABA, baclofen and 2-hydroxy saclofen in a dose-dependent manner. Furthermore, the purified GABAB receptor showed approximately 2800-fold purification as compared with the original solubilized fraction and possessed the specific binding activity of 17.68 p mol/mg of protein. This binding consisted of a single binding site with a dissociation constant of 64.4 nM. The present results indicate that affinity column chromatographic procedures using baclofen-coupled epoxy-activated Sepharose 6B are suitable for the partial purification of GABAB receptor from cerebral tissues.     

Expression,refolding and purification of a human interleukin-17A variant

A human interleukin-17A (IL-17A) variant was overexpressed in Escherichia coli BL21 (DE3) under the control of a T₇ promoter. The resulting insoluble inclusion bodies were isolated and solubilized by homogenization with 6 M guanidine HCl. The denatured recombinant human IL-17A variant was refolded in 20 mM Tris–HCl, pH 9.0, 500 mM arginine, 500 mM guanidine HCl, 15% glycerol, 1 mM cystamine, and 5 mM cysteine at 2–8 °C for 40 h. The refolded IL-17A variant was subsequently purified using a combination of cation-exchange, reversed-phase and fluoroapatite chromatography. The final purified product was a monodisperse and crystallizable homodimer with a molecular weight of 30,348.3 Da. The protein was active in both receptor binding competition assay and IL-17A-dependent biological activity assay using human dermal fibroblasts.     

Overexpression and purification of the three components of the Enterobacter aerogenes AcrA-AcrB-TolC multidrug efflux pump

The tripartite AcrA-AcrB-TolC system is the major efflux pump of the nosocomial pathogen Enterobacter aerogenes. AcrA is a trimeric periplasmic lipoprotein anchored in the inner membrane, AcrB is an inner membrane transporter and TolC is a trimeric outer membrane channel. In order to reconstitute the AcrA-AcrB-TolC system of E. aerogenes in artificial membranes, we overexpressed and purified the three proteins. The E. aerogenes acrA, acrB and tolC open reading frames were individually inserted in the expression vector pET24a(+), in frame with a sequence coding a C-terminal hexahistidine tag to allow purification by INAC (Immobilized Nickel Affinity Chromatography). The mature AcrA-6His was overproduced in a soluble form in the cytoplasm of Escherichia coli BL21(DE3). AcrA-6His was purified under native conditions in two steps using INAC and gel permeation chromatography. We obtained about 25 mg of 97% pure AcrA-6His per liter of culture. AcrB-6His was solubilized from the membrane fraction of E. coli C43(DE3) in 300 mM NaCl, 5% Triton X-100 and purified in one step by INAC. The AcrB-6His enriched fraction was eluted with 100 mM imidazole. The final yield was 1-2 mg of 95% pure AcrB-6His per liter of culture. The membrane fraction of E. coli BL21(DE3)pLysS containing TolC-6His was first treated with 2% Triton X-100, 30 mM MgCl(2) to solubilize the inner membrane proteins. After ultracentrifugation, the pellet was treated with 5% Triton X-100, 5 mM EDTA to solubilize the outer membrane proteins. Approximately 5 mg of 95% pure TolC-6His trimers per liter of culture was purified by INAC.     

Chymotryptic heavy meromyosin from gizzard myosin: a proteolytic fragment with the regulatory properties of the intact myosin.

Arginine deiminase (EC 3.5.3.6) from $\text{Mycoplasma}\text{arthritidis}$ is a dimeric enzyme. Velocity centrifugation in 6 M guanidine HCl and peptide mapping of the BrCN fragments suggest that the subunits are identical. The reaction of one out of four sulfhydryl groups with 0.3 mM 5,5′-dithiobis-(2-nitrobenzoic acid) has a half-life of about 30 min in 2 M guanidine HCl at 15°, pH 8. The enzyme is irreversibly inhibited by 1 mM formamidinium ion within 1 min. Inactivation by this affinity label is resolvable into two concurrent first-order reactions in the presence of guanidinium ion; the fraction of enzyme which reacts at the faster rate is about 50%. These results are interpreted as evidence for two catalytic subunits which differ in conformation.     

Affinity purification of antibodies by using Ni2+-resins on which inclusion body-forming proteins are immobilized

Bacterially expressed recombinant proteins are widely used for producing specific antibodies. Unfortunately, many recombinant proteins are recovered as insoluble materials, so-called inclusion bodies. Inclusion bodies are rather advantageous from a point of view of immunogens because fairly pure proteins can be feasibly extracted from the inclusion bodies. However, we encounter a problem with an insoluble protein when we make an antigen-immobilized column for affinity purification of antibodies because we need a soluble protein in usual immobilization methods. Histidine-tagged proteins can be bound to Ni(2+)-resins in buffer containing 6M guanidine-HCl, in which most insoluble proteins are solubilized. Taking advantage of this feature, we have successfully purified antigen-specific antibodies by directly using Ni(2+)-resins onto which denatured proteins are bound.     

Characterization of a biologically active extracellular domain of fibroblast growth factor receptor 1 expressed in Escherichia coli.

The functional features of a recombinant fibroblast growth factor (FGF) receptor (FGF-R) were investigated by expressing at high level in Escherichia coli a soluble non-glycosylated form of FGF-R1. The extracellular domain of the mature protein (XC-FGF-R), comprising the first 356 amino acids, was purified from a large-scale fermentation. After cell lysis, the protein was quantitatively found in the pellet. XC-FGF-R was solubilized using guanidine/HCl and allowed to refold using two dialysis steps. The refolded protein was obtained in a homogeneous form after ammonium sulphate precipitation and gel-filtration chromatography. The soluble receptor had the ability to form a complex with recombinant human basic FGF (rhbFGF) in solution, as demonstrated by immunoprecipitation with anti-(FGF-R) serum. Formation of a rhbFGF/XC-FGF-R complex was visualized by cross-linking experiments. Quantitative binding experiments with the XC-FGF-R immobilized on Affi-Gel resin showed high binding affinity for 125I-bFGF (Kd = 5-10 nM). Purified XC-FGF-R inhibited binding of 125I-bFGF to its high-affinity receptors on baby hamster kidney cells. These data suggest that glycosylation of the FGF-R is not necessary for its ligand-binding activity. The use of an E. coli expression system resulted in the efficient production of a soluble receptor in a form suitable for ligand/receptor structural studies and screening of new potential agonists and antagonists of angiogenesis. These results indicate that E. coli can be used for the production of complex molecules such as Ig-like receptors.     

Differential down-regulation of protein kinase C selectively affects IgE-dependent exocytosis and inositol trisphosphate formation.

Recombinant Mu gam gene protein (Mu GAM) synthesized in Escherichia coli accumulates in the form of insoluble inclusion bodies which, after cell lysis and low-speed centrifugation, can be recovered in the pellet fraction. This property was utilized in a purification procedure for Mu GAM based on guanidine hydrochloride denaturation-renaturation followed by a single DEAE-cellulose chromatographic step. The purified Mu GAM was shown by nitrocellulose-filter-binding experiments to bind with high affinity to linear double-stranded DNA and more weakly to supercoiled and single-stranded forms. Mu GAM protects linear DNA from degradation by a variety of exonucleases, but only weakly inhibits endonuclease activity. These results are in accord with a model of Mu GAM conferring protection from exonuclease activity by binding to the ends of DNA.     

Production and purification of refolded recombinant human IL-7 from inclusion bodies

A recombinant form of human rhIL-7 was overexpressed in Escherichia coli HMS174 (DE3) pLysS under the control of a T7 promoter. The resulting insoluble inclusion bodies were separated from cellular debris by cross-flow filtration and solubilized by homogenization with 6 M guanidine HCl. Attempts at refolding rhIL-7 from solubilized inclusion bodies without prior purification of monomeric, denatured rhIL-7 were not successful. Denatured, monomeric rhIL-7 was therefore initially purified by size-exclusion chromatography using Prep-Grade Pharmacia Superdex 200. Correctly folded rhIL-7 monomer was generated by statically refolding the denatured protein at a final protein concentration of 80-100 microg/ml in 100 mM Tris, 2mM EDTA, 500 mM L-arginine, pH 9.0, buffer with 0.55 g/l oxidized glutathione at 2-8 degrees C for at least 48 h. The refolded rhIL-7 was subsequently purified by low-pressure liquid chromatography, using a combination of hydrophobic interaction, cation-exchange, and size-exclusion chromatography. The purified final product was >95% pure by SDS-PAGE stained with Coomassie brilliant blue, high-pressure size-exclusion chromatography (SEC-HPLC), and reverse-phase HPLC. The endotoxin level was <0.05 EU/mg. The final purified product was biologically active in a validated IL-7 dependent pre-B-cell bioassay. In anticipation of human clinical trials, this material is currently being evaluated for safety and efficacy in non-human primate toxicology studies.     

Comparative binding of lactate dehydrogenase to mitochondrial fractions

Beef liver mitochondrial fraction showed LDH activity (1.76 +/- 0.25 U/g pellet). Sixty seven% of the initial mitochondrial pellet LDH activity (almost M4 isoenzyme) was released when suspended in NaCl 0.15 M. When the washed particles were sonicated in a 0.15 M NaCl medium, the solubilized LDH activity (all five isoenzymes as cytosoluble fraction) was 5-fold higher than the initial pellet activity. The different isoenzymatic composition of intramitochondrial and externally bound forms of the enzyme should be taken into account when investigating the physiological role of intramitochondrial LDH. Beef liver cytosoluble LDH (very little content of M4 isoenzyme) showed no affinity for the beef liver mitochondrial fraction but purified M4-LDH isoenzyme was able to bind to the particulate fraction from the same source. This suggests an isoenzyme specificity for the interaction. The maximum amount of cytosoluble LDH bound to the mitochondrial fraction depends on the enzyme and the particulate fraction source. Therefore, binding capacity to the mitochondrial fraction depends not only on the net charge of LDH isoenzymes, which play a predominant role in the binding, but also on individual characteristics of the LDH isoenzymes and mitochondrial fractions from different sources. This suggests that electrostatic forces are not the only ones involved in the binding process.     

Expression and purification of recombinant tung tree diacylglycerol acyltransferase 2

Diacylglycerol acyltransferases (DGATs) esterify sn-1,2-diacylglycerol with a long-chain fatty acyl-CoA, the last and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. At least 74 DGAT2 sequences from 61 organisms have been identified, but the expression of any DGAT2 as a partial or full-length protein in Escherichia coli had not been reported. The main objective of this study was to express and purify recombinant DGAT2 (rDGAT2) from E. coli for antigen production with a minor objective to compare rDGAT2 expression in yeast. A plasmid was engineered to express tung tree DGAT2 fused to maltose binding protein and poly-histidine (His) affinity tags. Immunoblotting showed that rDGAT2 was detected in the soluble, insoluble, and membrane fractions. The rDGAT2 in the soluble fraction was partially purified by amylose resin, nickel-nitrilotriacetic agarose (Ni-NTA) beads, and tandem affinity chromatography. Multiple proteins co-purified with rDGAT2. Size exclusion chromatography estimated the size of the rDGAT2-enriched fraction to be approximately eight times the monomer size. Affinity-purified rDGAT2 fractions had a yellow tint and contained fatty acids. The rDGAT2 in the insoluble fraction was partially solubilized by seven detergents with SDS being the most effective. Recombinant DGAT2 was purified to near homogeneity by SDS solubilization and Ni-NTA affinity chromatography. Mass spectrometry identified rDGAT2 as a component in the bands corresponding to the monomer and dimer forms as observed by SDS-PAGE. Protein bands with monomer and dimer sizes were also observed in the microsomal membranes of Saccharomyces cerevisiae expressing hemagglutinin-tagged DGAT2. Nonradioactive assay showed TAG synthesis activity of DGAT2 from yeast but not E. coli. The results suggest that rDGAT2 is present as monomer and dimer forms on SDS-PAGE, associated with other proteins, lipids, and membranes, and that post-translational modification of rDGAT2 may be required for its enzymatic activity and/or the E. coli protein is misfolded.     

Lactate dehydrogenase associated with the mitochondrial fraction and with a mitochondrial inhibitor--I. Enzyme binding to the mitochondrial fraction

Rabbit skeletal muscle mitochondrial fraction shows LDH activity (212 +/- 43 U/g pellet). The majority of the mitochondrial enzyme was solubilized by washing with 0.15 M NaCl, pH 6, or by ultrasonic treatment in the same medium. It was also solubilized on increasing the ionic strength and the pH of the medium. Cytosoluble LDH was observed to bind in vitro to the particulate fraction and the enzyme bound was a sigmoidal function of the amount of soluble enzyme added. The bound enzyme is less active than the soluble one. Kinetically, active mitochondrial fraction or in vitro bound enzyme showed non-hyperbolic behavior which is different from the bi-bi sequential-ordered type mechanism of the soluble enzyme.     

Expression, single-step purification, and matrix-assisted refolding of a maize cytokinin glucoside-specific beta-glucosidase.

Availability of highly purified native beta-glucosidase Zm-p60.1 in milligram quantities was a basic requirement for analysis of structure-function relationships of the protein. Therefore, Zm-p60.1 was overexpressed to high levels as a fusion protein with a hexahistidine tag, (His)(6)Zm-p60.r, in Escherichia coli, resulting, however, in accumulation of most of the protein in insoluble inclusion bodies. Native (His)(6)Zm-p60.r was then purified either from the bacterial lysate soluble fraction or from inclusion bodies. In the first case, a single-step purification under native conditions based on immobilized metal affinity chromatography (IMAC) was developed. In the second case, a single-step purification protocol under denaturing conditions followed by IMAC-based matrix-assisted refolding was elaborated. The efficiency of the native protein purification from soluble fraction of bacterial homogenate was compared to the feasibility of purification and renaturation of the protein from inclusion bodies. Gain of authentic biological activity and quaternary structure after the refolding process was confirmed by K(m) determination and electrophoretic mobility under native conditions. The yield of properly refolded protein was assessed based on the specific activity of the refolded product.