High-efficient renaturation of immobilized recombinant C-terminal fragment of human alpha-fetoprotein

C-terminal fragment of a human oncofetal alpha-fetoprotein (AFP) may be used in targeted cytostatics delivery to malignant cells of many tumors. AFP fragment (from 404 to 595 amino acids residues of a full-sized protein) was cloned and produced in Escherichia coli cells, BL21 strain (DE3) in the form of inclusion bodies. To obtain a functionally active protein, is it necessary to renature the protein. The renaturation procedure of the AFP third domain (rAFP3D) is considerably complicated by the fact that the protein is hydrophobic and contains a large number of S-S bonds. A renaturation technique of rAFP3D immobilized on silicic metal chelate resin has been developed. The yield of renatured C-terminal fragment was no less than 60% with purity on the order of 98%. The developed technique has been applied for the first time for hydrophobic protein with a large number of S-S bonds. The approach can be applied for efficient renaturation of other hydrophobic proteins with a large number of disulfide bonds for scientific and practical purposes.     

Synthesis and characterization of a recombinant fragment of human alpha-fetoprotein with antigenic selectivity versus albumin

A DNA sequence coding for human alpha-fetoprotein amino acid sequence 38-119 was synthesized and cloned in a bacterial expression vector. The alpha-fetoprotein sequence was selected as the least homologous to albumin, since the two proteins have an overall amino acid identity of approximately 38%. A chimeric protein was obtained which was purified by preparative electrophoresis and characterized in its primary structure by fast atom bombardment mass spectometry. About 70% of the alpha-fetoprotein sequence was physically mapped and found to correspond to the amino acids encoded in the synthetic gene. The use of this recombinant protein allowed the selection of monoclonal antibodies recognizing both the recombinant fragment and native alpha-fetoprotein. These antibodies should allow the development of an immunoassay for alpha-fetoprotein with absolute selectivity versus albumin. This might result in more sensitive clinical determinations, avoiding the possibility of cross-reactions.     

Purification and characterization of recombinant extracellular domain of human HER2 from Escherichia coli

The human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor (EGFR) family, and it plays an important role in the development of many human adenocarcinomas. The extracellular domain (ECD) of HER2 is an ideal target for therapeutic approaches. In order to obtain large quantities of active HER2 ECD protein for biochemical and structural analysis and for detecting anti-HER2 ECD antibodies in serum, a systematic assessment of optimal parameters for the refolding of the glutathione S-transferase (GST) fusion protein was carried out. After the GST-HER2 ECD inclusion bodies were solubilized with denaturation buffer containing 8M urea, an approach was then used to optimize refolding parameters. This approach utilized dilution of denatured and reduced GST-HER2 ECD into different refolding buffers using orthogonal design method. Optimal refolding was obtained in an alkaline buffer containing reduced and oxidized glutathione, and subsequent incubation at 4 degrees C for 24h. After purification with glutathione Sepharose 4B and PreScission protease cleavage of the fusion protein, 8.9mg of recombinant HER2 ECD was obtained from 1L of Escherichia coli. Rabbit polyclonal antibodies against HER2 ECD were obtained. The purified protein was found to be immunogenic and useful for immunodiagnostic studies of serum HER2 ECD and its antibodies by using enzyme-linked immunosorbent assay (ELISA).     

Purification and structural characterization of the central hydrophobic domain of oleosin

The oil bodies of rapeseeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with abundant structural alkaline proteins termed oleosins and some other minor proteins. Oleosins are unusual proteins because they contain a 70-80-residue uninterrupted nonpolar domain flanked by relatively polar C- and N-terminal domains. Although the hydrophilic N-terminal domain had been studied, the structural feature of the central hydrophobic domain remains unclear due to its high hydrophobicity. In the present study, we reported the generation, purification, and characterization of a 9-kDa central hydrophobic domain from rapeseed oleosin (19 kDa). The 9-kDa central hydrophobic domain was produced by selectively degrading the N and C termini with enzymes and then purifying the digest by SDS-PAGE and electroelution. We have also reconstituted the central domain into liposomes and synthetic oil bodies to determine the secondary structure of the domain using CD and Fourier transform infrared (FTIR) spectroscopy. The spectra obtained from CD and FTIR were analyzed with reference to structural information of the N-terminal domain and the full-length rapeseed oleosin. Both CD and FTIR analysis revealed that 50-63% of the domain was composed of beta-sheet structure. Detailed analysis of the FTIR spectra indicated that 80% of the beta-sheet structure, present in the central domain, was arranged in parallel to the intermolecular beta-sheet structure. Therefore, interactions between adjacent oleosin proteins would give rise to a stable beta-sheet structure that would extend around the surface of the seed oil bodies stabilizing them in emulsion systems. The strategies used in our present study are significant in that it could be generally used to study difficult proteins with different independent structural domains, especially with long hydrophobic domains.     

Purification of human alpha-fetoprotein

By employing complex and highly specialized immunochemical methods, several investigators have achieved purification of human α-fetoprotein (AFP) found in fetal serum and/or sera of patients with hepatoma. The present report describes a simpler method which results in the isolation of homogeneous preparation of AFP from human cord serum. AFP was purified by sequential use of Affi-Gel Blue affinity, DE-52 diethylaminoethyl cellulose ion-exchange, immunoadsorption with anti-albumin covalently coupled to Sepharose 4B, and Sephacryl S-200 molecular sieve chromatographic techniques. The homogeneity of the purified AFP was established by subjecting it to polyacrylamide gel electrophoresis, analytical isoelectric focusing, molecular sieve chromatography and immunological techniques. The purified AFP has a molecular weight of approximately 68,000 as determined by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate and molecular sieve chromatography, and upon isoelectric focusing yielded a single band pI = 4.8. In addition, the purified AFP gives a single precipitin line when tested against rabbit antiserum to whole human hepatoma serum proteins, and no line(s) of precipitin when tested against rabbit antiserum to normal serum proteins.     

Preparation and properties of a single-sited fragment from the C-terminal domain of human transferrin

A single-sited iron-binding fragment of human transferrin has been obtained by thermolysin cleavage of the protein, selectively loaded with iron in the C-terminal binding site, in a urea-containing buffer. The fragment contains carbohydrate, and hence derives from the C-terminal half of transferrin. Its metal-binding site accepts Fe3+ and Cu2+ with bicarbonate as accompanying anion, but only Fe3+ with oxalate as anion. EPR spectroscopic properties of the fragment are similar to those of the corresponding site in the intact protein. However, iron-binding by the fragment is weaker than by the C-terminal site of the intact protein, particularly at low pH, suggesting that overall as well as local protein conformation influences the metal-binding functions of the site.     

Expression and characterization of a recombinant Fab fragment derived from an anti-human alpha-fetoprotein monoclonal antibody

Alpha-fetoprotein (AFP) is a well-known molecular marker indicating the development of cancer as well as fetal abnormalities such as open neural tube defects. Accordingly the measurement of serum AFP is important for the diagnosis of hepatocellular carcinoma (HCC) and other abnormalities. Monoclonal antibodies (McAb) to AFP were produced to develop an immunoassay kit, and to study the possibility of an antibody (Ab) therapy. The immunoglobulin genes were cloned from hybridoma cells, and expressed in E. coli as an Fab soluble into a culture medium. The Fab of anti-AFP McAb exhibited binding to AFP and similar affinity compared to the original IgG. This recombinant antibody can be studied further for in vivo imaging and immunotherapeutics.     

A structural characterization of the interactions between titin Z-repeats and the alpha-actinin C-terminal domain

Titin and alpha-actinin, two modular muscle proteins, are with actin the major components of the Z-band in vertebrate striated muscles where they serve to organize the antiparallel actin filament arrays in adjacent sarcomeres and to transmit tension between sarcomeres during activation. Interactions between titin and alpha-actinin have been mainly localized in a 45-amino acid multiple motif (Z-repeat) in the N-terminal region of titin and the C-terminal region of alpha-actinin. In this study, we provide the first quantitative characterization of alpha-actinin-Z-repeat recognition and dissect the interaction to its minimal units. Different complementary techniques, such as circular dichroism, calorimetry, and nuclear magnetic spectroscopy, were used. Two overlapping alpha-actinin constructs (Act-EF34 and Act-EF1234) containing two and four EF-hand motifs, respectively, were produced, and their folding properties were examined. Complex formation of Act-EF34 and Act-EF1234 with single- and double-Z-repeat constructs was studied. Act-EF34 was shown quantitatively to be necessary and sufficient for binding to Z-repeats, excluding the presence of additional high-affinity binding sites in the remaining part of the domain. The binding affinities of the different Z-repeats for Act-EF34 range from micromolar to millimolar values. The strongest of these interactions are comparable to those observed in troponin C-troponin I complexes. The binding affinities for Act-EF34 are maximal for Zr1 and Zr7, the two highly homologous sequences present in all muscle isoforms. No cooperative or additional contributions to the interaction were observed for Z-repeat double constructs. These findings have direct relevance for evaluating current models of Z-disk assembly.     

Purification and characterization of recombinant human prostacyclin synthase

Prostacyclin synthase (PGIS), which catalyzes the conversion of prostaglandin (PG) H(2) to prostacyclin (PGI(2)), is a member of the cytochrome P-450 (P450) superfamily, CYP8A1. To study the enzymatic and protein characteristics of human PGIS, the enzyme was overexpressed in Spodoptera frugiperda 21 (Sf21) cells using the baculovirus expression system. PGIS was expressed in the microsomes of the infected Sf21 cells after culture in 5 microg/ml hematin-supplemented medium for 72 h. The holoenzyme was isolated from the solubilized microsomal fraction by calcium phosphate gel absorption and purified to homogeneity by DEAE-Sepharose and hydroxyapatite column chromatography. The K(m) and V(max) values of the purified human PGIS for PGH(2) were 30 microM and 15 micromol/min/mg of protein at 24 degrees C, respectively. The optical absorption and EPR spectra of the enzyme revealed the characteristics of a low-spin form of P450 in the oxidized state. The carbon monoxide-reduced difference spectrum, however, exhibited a peak at 418 nm rather than 450 nm. The addition of a PGH(2) analogue, U46619, to the enzyme produced an oxygen-ligand type of the difference spectrum with maximum absorption at 407 nm and minimum absorption at 430 nm. Treatment with another PGH(2) analogue, U44069, produced a peak at 387 nm and a trough at 432 nm in the spectrum (Type I), while treatment with tranylcypromine, a PGIS inhibitor, produced a peak at 434 nm and a trough at 412 nm (Type II). A Cys441His mutant of the enzyme possessed no heme-binding ability or enzyme activity. Thus, we succeeded in obtaining a sufficient amount of the purified recombinant human PGIS from infected insect cells for spectral analyses that has high specific activity and the characteristics of a P450, indicating substrate specificity.     

Preliminary X-ray crystallography studies of recombinant human interleukin-1 alpha. Purification and structural characterization

Human interleukin-1 alpha, cloned and expressed in E. coli, has been purified and structurally characterized by various physiochemical methods, including mass spectrometry. The recombinant protein has been crystallized by the hanging drop vapor diffusion method using dimethyl sulfoxide as the precipitating agent. The space group is P2(1)2(1)2(1). Unit cell dimensions are a = 44.1, b = 57.1, and c = 61.7 A and alpha = beta = gamma = 90 degrees. The crystals diffract to beyond 1.7 A and are suitable for high resolution data collection. Native diffraction data were collected. Screens for heavy atom derivatives have been initiated.     

Purification and structural characterization of human ERp29

ERp29 is a major resident of the endoplasmic reticulum (ER) and is postulated to play an important molecular chaperone role in most animal cells. Human ERp29 was isolated to homogeneity in high yield by using a bacterial expression system. Its secondary structure was studied by circular dichroism (CD), Fourier transformed infrared spectroscopy (FTIR) and Raman spectroscopy and it was found that human ERp29 comprises significant alpha-helical structure. The details of its temperature-induced conformational changes was studied by CD and FTIR for the first time, revealing that the protein is stable below 50 degrees C and has two distinct structural transitions between 50 degrees C and 70 degrees C. This may shed light on ERp29's inability to protect substrate proteins against thermal aggregation.     

Subcloning,expression, purification,and characterization of recombinant human leptin-binding domain

A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced by SDS-PAGE, with over 95% monomeric protein. The purified leptin-binding domain (LBD) exhibited the predicted beta structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins. The binding kinetics, assayed by surface plasmon resonance methodology, showed respective k(on) and k(off) values (mean +/- S.E.) of 1.20 +/- 0.23 x 10(-5) mol(-1) s(-1) and 1.85 +/- 0.30 x 10(-3) s(-1) and a K(d) value of 1.54 x 10(-8) m. Similar results were achieved with conventional binding experiments. LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD.human leptin complex. Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding.     

Purification and structural characterization of recombinant rat gamma-interferon from Escherichia coli

An efficient method has been developed for the purification of recombinant rat gamma-interferon (rat rIFN-gamma). The procedure involves extraction of the Escherichia coli cell paste with 6 M guanidine-HCl (GuHCl), adsorption of the rat rIFN-gamma onto C8 alkyl-bonded silica, and elution with 50% propanol. The protein is essentially pure at this step, but is quantitatively precipitated by threefold dilution with aqueous buffer at pH 8.5. The precipitate is then dissolved with 6 M GuHCl in a buffer containing 0.05%. Tween-80 to about 0.3 mg/ml and dialyzed against the same buffer. The rat rIFN-gamma, which remains soluble on dialysis is again precipitated by dialysis against ammonium sulfate at 80% saturation. This final precipitate is readily soluble in 0.1 M ammonium acetate buffer, pH 8.5. The preparation is fully active and possesses a specific activity of 2-6 X 10(6) units/mg. The recoveries ranged from 50 to 85% in several experiments. The sequence of 20 amino acid residues from the NH2-terminus of the protein was determined using an automated sequencer and was found to agree with that deduced from the cDNA sequence.     

Purification and characterization of human recombinant interleukin-1 beta

A human interleukin-1 (IL-1) beta cDNA was cloned, and the region coding for the mature protein was expressed in Escherichia coli. The 17-kDa biologically active product was purified in 40% yield to apparent homogeneity, without chaotropes, from the soluble fraction of sonicated cell lysates. The recombinant IL-1 beta was characterized by amino acid analysis, NH2- and COOH-terminal sequence analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectroscopy, and biological assay. Specific biological activity was 4.6 X 10(8) units/mg in a co-mitogenic IL-2 induction assay using cultured EL-4 T-lymphocytes. The molar extinction coefficient was determined to be 10,300 cm-1 M-1 at 280 nm. NH2-terminal sequence analysis revealed that 70% of the product begins with the Ala corresponding to the NH2 terminus of the natural protein, while 30% begins with the following Pro. No initiator Met was observed. Both of the sulfhydryl groups are reactive to Ellman's reagent and to iodoacetamide under nonreducing conditions, indicating that the Cys residues do not form disulfide bonds. S-Carboxamidomethyl-Cys-rIL-1 beta retained biological activity in the IL-2 induction assay. Circular dichroism suggested an extensive beta sheet structure for rIL-1 beta.     

Expression and structural characterization of the recombinant human doppel protein

The doppel protein (Dpl) is a newly recognized prion protein (PrP)-like molecule encoded by a novel gene locus, prnd, located on the same chromosome as the PrP gene. To study the structural features of Dpl, we have expressed recombinant human Dpl corresponding to the putative mature protein domain (residues 24-152) in Escherichia coli. The primary structure of the recombinant Dpl 24-152 was characterized using gel electrophoresis, N-terminal Edman sequencing, matrix-assisted laser desorption ionization mass spectrometry, and electrospray ionization mass spectrometry. Dpl 24-152 was shown to contain two disulfide bonds (Cys94-Cys145 and Cys108-Cys140). The secondary structure of Dpl was analyzed using far-UV circular dichroism spectroscopy. Dpl 24-152 was found to be an alpha-helical protein having a high helical content (40%). Dpl 24-152 exhibited characteristics of a thermodynamically stable protein that undergoes reversible and cooperative thermal denaturation. In addition, Dpl was found to be soluble and sensitive to proteinase K digestion. Therefore, Dpl 24-152 possesses biochemical properties similar to those of recombinant PrP. This study provides knowledge about the molecular features of human Dpl that will be useful in further investigation into its normal function and the role it may play in neurodegenerative diseases.     

Purification and characterization of recombinant human liver glycolate oxidase

Glycolate oxidase, an FMN-dependent peroxisomal oxidase, plays an important role in plants, related to photorespiration, and in animals, where it can contribute to the production of oxalate with formation of kidney stones. The best studied plant glycolate oxidase is that of spinach; it has been expressed as a recombinant enzyme, and its crystal structure is known. With respect to animals, the enzyme purified from pig liver has been characterized in detail in terms of activity and inhibition, the enzyme from human liver in less detail. We describe here the purification and initial characterization of the recombinant human glycolate oxidase. Its substrate specificity and the inhibitory effects of a number of anions are in agreement with the properties expected from previous work on glycolate oxidases from diverse sources. The recombinant enzyme presents an inhibition by excess glycolate and by excess DCIP, which has not been documented before. These inhibitions suggest that glycolate binds to the active site of the reduced enzyme, and that DCIP also has affinity for the oxidized enzyme. Glycolate oxidase belongs to a family of l-2-hydroxy-acid-oxidizing flavoenzymes, with strongly conserved active-site residues. A comparison of some of the present results with studies dealing with other family members suggests that residues outside the active site influence the binding of a number of ligands, in particular sulfite.     

Purification and characterization of recombinant human parathyroid hormone-related protein

Full-length human parathyroid hormone-related protein (PTHrP-(1-141] as well as a carboxyl-terminal shortened form (PTHrP-(1-108] have been expressed from recombinant DNA-derived clones. These proteins were expressed in Escherichia coli as fusion proteins so that cyanogen bromide cleavage yields the desired product. Both proteins were purified and then characterized by sodium dodecyl sulfate gel electrophoresis, amino-terminal amino acid sequencing, peptide mapping, and mass spectral analysis. Recombinant PTHrP-(1-141), PTHrP-(1-108), synthetic PTHrP-(1-34), and naturally derived PTHrP are all equipotent in the stimulation of cyclic AMP levels in the osteoblast-like cell line UMR 106-01. However, PTHrP-(1-141) and -(1-108) are two to four times more active than PTHrP-(1-34) in the stimulation of plasminogen activator activity from this cell line. PTHrP-(1-141) reacts equipotently with PTHrP-(1-34) in a radioimmunoassay using an antiserum prepared against PTHrP-(1-34). PTHrP-(1-141), -(1-108), and -(1-84) were used as PTHrP-specific mobility standards on sodium dodecyl sulfate gel electrophoresis to determine the approximate length of two forms of naturally derived PTHrP. The data show that PTHrP purified from the lung tumor cell line BEN contains a major form of about 108 amino acids and another form of about 141 amino acids.     

Purification and partial characterization of recombinant human differentiation-stimulating factor

Recombinant human differentiation-stimulating factor (rhD-factor) has been isolated to greater than 95% purity from Chinese hamster ovary cells. RhD-factor is a glycoprotein with an apparent molecular weight of 45.6 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On gel filtration in 6 M guanidine-hydrochloride, rhD-factor elutes with an apparent molecular weight of 21.5 kDa; it elutes with an apparent molecular weight of 44.8 kDa under neutral pH (native) conditions. The amino-terminal sequence (12 residues) is consistent with the expected sequence derived from the genomic DNA sequence. Recombinant D-factor is heavily glycosylated with 30% by weight neutral sugar and 12% sialic acid. The ED50 for rhD-factor was 0.25 ng/ml. Trifluoromethanesulfonic acid-deglycosylated rhD-factor has a biological activity comparable to that of the native recombinant protein (ED50 = 0.40 ng/ml). The biological activity of rhD-factor was stable at pH 1 for 40 h, in 6 M guanidine-HCl containing buffers with or without reducing agent, and in 1% SDS. Carboxymethylation of D-factor after reduction totally destroyed biological activity.