Practical aspects of fluorescent staining for proteomic applications.

SYPRO Orange and SYPRO Ruby staining methods, modified for use with large-format two dimensional (2-D) gels, are compared to the manufacturer's recommended protocols to determine sensitivity and reproducibility of the new methods. This study examines the critical aspects of fixation, washing, and staining to develop an optimized fluorescent staining method. It was determined that careful control of sodium dodecyl sulfate (SDS) levels and pH in the gel was critical for successful staining with SYPRO Orange. Overnight fixation in 40% ethanol/2% acetic acid/0.0005% SDS preserved protein content, eliminated ampholyte-generated staining artifacts, and had no detrimental effects on staining. Three one-hour washes in 2% acetic acid/0.0005% SDS, followed by staining with SYPRO Orange diluted 1:5,000 with washing solution for 3 or more hours, produced high sensitivity, low background images using a STORM 860 laser scanner. Gels viewed two years after staining showed no significant changes with respect to the initial protein patterns, and allowed successful mass spectrometric postgel characterization of protein spots. Protocol changes applied to SYPRO Ruby staining improved the contrast of STORM 860-generated images, but had little impact on staining sensitivity. A comparison of the cost benefits of staining with SYPRO Orange vs. SYPRO Ruby is also discussed.

Multiple conformations of a human interleukin-3 variant.

Interleukin-3 (IL-3) is a cytokine that stimulates the proliferation and differentiation of hematopoietic cells. The hyperactive hIL-3 variant SC-55494 was shown to have at least two major conformations by high-resolution NMR spectroscopy. Mutants of SC-55494 were constructed in which alanine was substituted for proline in order to test the hypothesis that proline cis-trans isomerization is the source of the observed conformational heterogeneity, as well as to evaluate the effect of prolyl peptide bond configuration on biological activity. NMR spectra of four single proline-to-alamine mutants (P30A, P31A, P33A, and P37A) retain doubled resonances, while spectra of the double mutant P30A/P31A and the quadruple mutant P30A/P31A/P33A/ P37A are substantially free of heterogeneity. These observations suggest that the two major conformations in SC-55494 correspond to cis and trans isomers of either or both of the R29-P30 and P30-P31 peptide bonds. All six mutants had somewhat lower cell proliferative activity than SC-55494, with relative activities ranging from 40 to 80%. The P37A mutant has a binding affinity to the low-affinity IL-3 receptor alpha-subunit statistically equivalent to SC-55494, while P30A, P31A, and P33A each had about two-fold decreases, and P30A/P31A and P30A/P31A/P33A/P37A had four-fold decreases. These findings suggest an important role for the cis configuration of either or both of the R29-P30 and P30-P31 peptide bonds in IL-3 for optimal interaction with the receptor alpha-subunit.

Large-scale production and characterization of Bacillus thuringiensis subsp. tenebrionis insecticidal protein from Escherichia coli.

Bacillus thuringiensis subsp. tenebrionis insecticidal protein was produced in recombinant Escherichia coli and purified to near homogeneity to provide quantities of protein for safety-assessment studies associated with the registration of transgenic potato plants. The 68-kDa protein is produced naturally by Bacillus thuringiensis subsp. tenebrionis by translation initiation at an internal initiation site in the native DNA sequence. The gene sequence specific for this truncated protein was expressed in E. coli strain JM 101 and fermented at the 1000-1 scale. The protein accumulated as insoluble inclusion bodies, and was purified by extraction at pH 10.8 with carbonate buffer, selective precipitation at pH 9.0, and differential centrifugation. No chromatography steps were required to produce over 50 g purified protein as a lyophilized powder with a purity greater than 95% and demonstrating full insecticidal activity against Colorado potato beetle larvae. The protein was further characterized to assure identity and suitability for use in safety-assessment studies.

Large-scale production of HIV-1 protease from Escherichia coli using selective extraction and membrane fractionation.

Human immunodeficiency virus type 1 (HIV-1) protease was expressed in Escherichia coli as a fusion protein with the N-terminal sequence of IGF-2. The protein accumulated in inclusion bodies as a 40:60 mixture of unprocessed fusion protein and processed protein. A simple purification procedure was developed that yielded 30-40 mg of active protease per liter of fermentation broth with a recovery of 30-40%. The purification process involved the selective extraction of HIV-1 protease from E. coli inclusion bodies with 50% acetic acid and fractional diafiltration to remove impurities and low-molecular-weight protease-related fragments. No chromatographic steps were employed, yet the HIV-1 protease produced by this procedure was greater than 95% pure by SDS-PAGE, reverse-phase HPLC, and N-terminal sequence analysis.

High level expression and refolding of mouse interleukin 4 synthesized in Escherichia coli.

Mouse Interleukin 4 is a 20-kDa glycoprotein, synthesized by activated T lymphocytes and mast cells, which regulates the growth and/or differentiation of a broad spectrum of target cells of the immune system, including B and T lymphocytes, macrophages, and hematopoietic progenitor cells. Using an inducible recA promoter and the g10-L ribosome-binding site, recombinant non-glycosylated interleukin 4 (IL-4) was expressed as 17% of total cellular protein in Escherichia coli inclusion bodies, as a reduced, inactive 14.5-kDa polypeptide. The protein was refolded and aggregates dissociated when three disulfide bonds were reformed by slowly decreasing the concentration of guanidine hydrochloride and cysteine. The oxidized monomer was purified to homogeneity by sequential ion-exchange and size exclusion chromatography. When compared with native IL-4, E. coli-derived IL-4 displayed an identical specific activity of 4-7 x 10(7) units/mg. This recombinant IL-4 contained a three-amino-acid NH2-terminal extension, which did not affect its biological activity. Purified biologically active protein consisted of three isoforms as shown by two-dimensional gel electrophoresis, with a pI greater than 9.0. These data suggest that neither glycosylation nor the NH2 terminus of mouse IL-4 play a critical role in contributing to its in vitro biological activity.

Renaturation and purification of human tissue factor pathway inhibitor expressed in recombinant E. coli.

Tissue factor pathway inhibitor is an inhibitor of the extrinsic coagulation pathway. Evaluation of the pharmacological effects of tissue factor pathway inhibitor in animal models has been limited by the high cost and low availability of mammalian tissue culture produced protein. In order to circumvent this obstacle, a 277-amino-acid nonglycosylated tissue factor pathway inhibitor variant possessing an N-terminal alanine was expressed in recombinant E. coli using the tac promoter expression system. High-level expression in recombinant E. coli resulted in the accumulation of ala-tissue factor pathway inhibitor in inclusion bodies. Active protein was produced by solubilization of the inclusion bodies in 8 M urea, purification of the full-length molecule by cation exchange chromatography, and renaturation in 6 M urea. Fractionation of crude refold mixtures using cation exchange chromatography yielded a purified nonglycosylated tissue factor pathway inhibitor possessing in vitro prothrombin time activity comparable to inhibitor purified from mammalian cell lines.

Purification and characterization of microbially expressed neomycin phosphotransferase II (NPTII) protein and its equivalence to the plant expressed protein.

The gene encoding neomycin phosphotransferase II (NPTII) has been used routinely as a selectable marker in the production of genetically engineered crops. To facilitate the safety assessment of this protein, the same coding sequence used for plant transformation was introduced into Escherichia coli to produce gram quantities of this protein. A unique, simple, rapid and efficient purification method was developed to purify thirty grams of NPTII protein. The microbially produced NPTII was shown to be chemically and functionally equivalent to the NPTII protein expressed in and purified from genetically engineered cotton seed, potato tubers and tomato fruit. Microbially produced and plant produced NPTII proteins have comparable molecular weights, immuno-reactivities, epitope structures, amino terminal amino acid sequences, biological activities and both lack glycosylation. Demonstrating the equivalence of NPTII protein from these sources establishes the validity of using the microbially produced NPTII to assess the safety of the NPTII protein produced in genetically engineered crops.

Safety assessment of the neomycin phosphotransferase II (NPTII) protein.

Two approaches were used to assess the safety of the NPTII protein for human consumption using purified E. coli produced NPTII protein that was shown to be chemically and functionally equivalent to the NPTII protein produced in genetically engineered cotton seed, potato tubers and tomato fruit. The NPTII protein was shown, as expected, to degrade rapidly under simulated mammalian digestive conditions. An acute mouse gavage study confirmed that the NPTII protein caused no deleterious effects when administered by gavage at a cumulative target dosage of up to 5000 mg/kg of body weight. This dosage correlates to at least a million fold safety factor relative to the average daily consumption of potato or tomato, assuming all the potatoes or tomatoes consumed contained the NPTII protein. These results, along with previously published information, confirm that ingestion of genetically engineered plants expressing the NPTII protein poses no safety concerns.

The glycoprotease of Pasteurella haemolytica A1 eliminates binding of myeloid cells to P-selectin but not to E-selectin.

HL-60 cells and neutrophils treated with the glycoprotease from Pasteurella haemolytica A1, an enzyme which is specific for O-sialoglycoproteins, were found to be incapable of binding P-selectin but still bound E-selectin. Comparative analysis of [35-S] cysteine labeled proteins from HL-60 cells by 2-dimensional electrophoresis indicated that two major proteins with M(r) 100 and 115 kd were significantly removed from cells which had been treated.

Functional analysis of protein N-myristoylation: metabolic labeling studies using three oxygen-substituted analogs of myristic acid and cultured mammalian cells provide evidence for protein-sequence-specific incorporation and analog-specific redistribution.

Covalent attachment of myristic acid (C14:0) to the NH2-terminal glycine residue of a number of cellular, viral, and oncogene-encoded proteins is essential for full expression of their biological function. Substitution of oxygen for methylene groups in this fatty acid does not produce a significant change in chain length or stereochemistry but does result in a reduction in hydrophobicity. These heteroatom-containing analogs serve as alternative substrates for mammalian myristoyl-CoA:protein N-myristoyltransferase (EC 2.3.1.97) and offer the opportunity to explore structure/function relationships of myristate in N-myristoyl proteins. We have synthesized three tritiated analogs of myristate with oxygen substituted for methylene groups at C6, C11, and C13. Metabolic labeling studies were performed with these compounds and (i) a murine myocyte cell line (BC3H1), (ii) a rat fibroblast cell that produces p60v-src (3Xsrc), or (iii) NIH 3T3 cells that have been engineered to express a fusion protein consisting of an 11-residue myristoylation signal from the Rasheed sarcoma virus (RaSV) gag protein linked to c-Ha-ras with a Cys----Ser-186 mutation. This latter mutation prevents isoprenylation and palmitoylation of ras. Two-dimensional gel electrophoresis of membrane and soluble fractions prepared from cell lysates revealed different patterns of incorporation of the analogs into cellular N-myristoyl proteins (i.e., protein-sequence-specific incorporation). In addition, proteins were identified that underwent redistribution from membrane to soluble fractions after incorporating one but not another analog (analog-specific redistribution). Comparable studies using the model RaSV-ras chimeric protein also demonstrated analog-specific differences in incorporation, varying from approximately 25% of the total RaSV-ras chimeric protein with 5-octyloxypentanoate to greater than 50% with 12-methoxydodecanoate. Modification by this latter compound was so extensive that the amount of membrane-associated N-myristoylated protein was decreased. Incorporation of each of the analogs caused a dramatic redistribution to the soluble fraction, comparable to that seen when myristoylation was completely blocked by mutating the protein's site of myristate attachment (glycine) to an alanine residue. The demonstration that these analogs differ in the extent to which they are incorporated and in their ability to cause redistribution of any single protein suggests that they may also have sufficient selectivity to be of potential therapeutic value.

Secretion of active kringle-2-serine protease in Escherichia coli.

Active human tissue plasminogen activator variant kringle-2-serine protease (K2 + SP domains; referred to as MB1004) was synthesized as a secreted protein in Escherichia coli, isolated, and characterized. MB1004 is a relatively large and complex protein, approximately 38 kDa in size and containing nine disulfide bonds. MB1004 without a pro region was secreted into the periplasm of E. coli by fusing the protein to the PhoA leader peptide expressed from the tac promoter. Approximately 1% (20 micrograms/L broth) of the secreted MB1004 was purified from E. coli homogenates as a soluble, active enzyme by using a combination of lysine and Erythrina inhibitor affinity chromatography. Purified MB1004 was monomeric and single-chain, and the N-terminus was identical with the predicted amino acid sequence. The specific activity of purified MB1004 from E. coli was compared against the equivalent recombinant material purified from mammalian cells that was naturally glycosylated (MB1004G) or deglycosylated after treatment with N-glycanase (MB1004N). Results from four different in vitro assays showed that MB1004 and MB1004N had similar activities. Both exhibited 4-12-fold higher specific activity than MB1004G in plasminogen activation assays. These results suggest that an inaccurate picture of specific activity can be obtained if the effects of glycosylation are not considered. By utilization of secretion in E. coli, nonglycosylated MB1004 was purified without in vitro refolding and was shown to be suitable for structure-function studies.

Characterization of glycosylated bovine placental lactogen and the effect of enzymatic deglycosylation on receptor binding and biological activity.

Bovine placental lactogen (bPL) is a glycoprotein hormone that has both somatogenic and lactogenic properties. Purified preparations of the hormone contain many isoforms that are separated by isoelectric focusing. The sequence for bPL contains one consensus site for an N-linked oligosaccharide and many potential sites for O-linked sugars. To determine whether the isoforms are the result of differences in glycosylation, the oligosaccharide portion of bPL was partially characterized. In addition, a number of the isoforms were isolated and enzymatically deglycosylated to determine the effect of O- and N-linked glycosylation on biological activity. Biological activity was assessed in a somatotropin radioreceptor assay and also in the Nb2 lymphoma lactogenic bioassay. The structure of N-linked oligosaccharide was found to be sialylated and triantennary and appeared to be the same for all of the different charge isomers. Compositional analysis suggested that O-linked oligosaccharides were also present. Treatment of the intact hormone with neuraminidase resulted in the loss of some, but not all, of the isoforms, suggesting that a large degree of the charge heterogeneity is due to posttranslational modifications unrelated to glycosylation. Enzymatic removal of N-linked oligosaccharides from native bPL resulted in a 1.2-2.3-fold increase in binding to the somatotropin receptor, whereas receptor binding was unaffected by enzymatic removal of O-linked oligosaccharide. Lactogenic activity was affected very little by the removal of either type of oligosaccharide. The data suggests that glycosylation of bPL may have a small effect on receptor specificity, but that overall its presence does not dramatically affect receptor binding or biological activity.

Crystallization of a chymotrypsin inhibitor from Erythrina caffra seeds.

Crystals of a chymotrypsin inhibitor from Erythrina caffra seeds have been grown out of lithium sulfate, by the hanging drop method of vapor diffusion. The crystals belong to the rhombohedral space group R32, with a = 67.2 A and alpha = 99.4 degrees, and diffract to 3 A resolution.

Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis.

Vascular permeability factor (VPF) is an Mr 40-kD protein that has been purified from the conditioned medium of guinea pig line 10 tumor cells grown in vitro, and increases fluid permeability from blood vessels when injected intradermally. Addition of VPF to cultures of vascular endothelial cells in vitro unexpectedly stimulated cellular proliferation. VPF promoted the growth of new blood vessels when administered into healing rabbit bone grafts or rat corneas. The identity of the growth factor activity with VPF was established in four ways: (a) the molecular weight of the activity in preparative SDS-PAGE was the same as VPF (Mr approximately 40 kD); (b) multiple isoforms (pI greater than or equal to 8) for both VPF and the growth-promoting activity were observed; (c) a single, unique NH2-terminal amino acid sequence was obtained; (d) both growth factor and permeability-enhancing activities were immunoadsorbed using antipeptide IgG that recognized the amino terminus of VPF. Furthermore, 125I-VPF was shown to bind specifically and with high affinity to endothelial cells in vitro and could be chemically cross-linked to a high-molecular weight cell surface receptor, thus demonstrating a mechanism whereby VPF can interact directly with endothelial cells. Unlike other endothelial cell growth factors, VPF did not stimulate [3H]thymidine incorporation or promote growth of other cell types including mouse 3T3 fibroblasts or bovine smooth muscle cells. VPF, therefore, appears to be unique in its ability to specifically promote increased vascular permeability, endothelial cell growth, and angio-genesis.

Purification and characterization of pituitary bovine somatotropin.

Bovine somatotropin (bST) has been isolated from pituitary glands and compared in a variety of chemical analyses and bioassays with somatotropin derived from recombinant Escherichia coli. Comparison of pituitary extracts and purified bST by Western blot analysis of two-dimensional gels suggested that the immunoreactive somatotropin species present in the extract were also present in the purified material, with no significant losses or degradation as a result of the purification method. NH2-terminal sequence analysis indicated the presence of equal quantities of Ala-Phe-Pro-Ala-Met-Ser-Leu-Ser- and Phe-Pro-Ala-Met-Ser-Leu-Ser- sequences. The Met-Ser-Leu-Ser-NH2-terminal sequence, a degradation product observed in NIH standard lots, was not detected. Assay of bioactivity in a bovine liver receptor-binding assay and in a female rat growth assay showed pituitary bST and recombinant methionyl-bovine somatotropin to be equipotent. Tryptic maps and sequence analysis of pituitary-derived somatotropin suggest the presence of isoaspartate derivatization at Asp128.

Identification, purification, and characterization of truncated forms of the human nerve growth factor receptor.

We report the presence of truncated forms of the nerve growth factor receptor (NGFRt) in the conditioned medium of the human melanoma cell line A875 and in human urine and amniotic fluid. Radioiodinated nerve growth factor (125I-NGF) specifically bound to NGFRt was chemically cross-linked. After immunoprecipitation, labeled receptor species were visualized by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NGFRts were purified from human adult male urine or a mixture of human amniotic fluid and infant urine by using a combination of either ion exchange chromatography (adult) or ammonium sulfate precipitation (infant) and immunoaffinity chromatography. Typical yields were about 1 microgram/liter of adult urine and 75 micrograms/liter of amniotic fluid/infant urine. The purified proteins, with molecular masses of 45, 40, and 35 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%), were confirmed to be NGFRts by amino-terminal sequencing and were designated NGFRt-1, NGFRt-2, and NGFRt-3, respectively. The isoelectric points of these three species ranged from 3.3 to 3.95 and displayed intraspecies heterogeneity; subsequently, amino acid residues covalently modified with sialic acid-containing carbohydrates were documented. The binding affinities of these species for nerve growth factor were comparable to that of the low affinity cell surface receptor. The potential to isolate milligram quantities of human NGFRts allows for model studies of the physicochemical structure of the intact receptor and the generation of polyclonal antibodies to study the biological functions of the NGF receptor.

Irreversible inhibition by acetaldehyde of cholecystokinin-induced amylase secretion from isolated rat pancreatic acini.

Acetaldehyde inhibited both amylase secretion induced by maximal concentrations (300 pM) of cholecystokinin octapeptide and the binding of radioiodinated cholecystokinin to receptors on isolated rat pancreatic acini. This inhibition was concentration dependent (10 mM to 1 M for amylase secretion and 100 mM to 1 M for binding). However, a correlation between the two inhibitory effects could not be obtained. Furthermore, the inhibitory effects were not reversible. Acetaldehyde did not alter the basal amylase secretion between 6 and 45 mM concentrations. However, 60, 100 and 300 mM acetaldehyde significantly decreased basal amylase secretion; no significant change in amylase secretion was observed at 600 mM and 1 M. Higher concentrations of acetaldehyde produced a 2- to 10-fold increase in basal amylase secretion. 51Cr release from prelabeled acini revealed no significant cell membrane damage between 10 and 600 mM acetaldehyde. These data suggest that acetaldehyde inhibition of cholecystokinin-induced amylase secretion is intracellularly mediated.

Purification and characterization of the membrane adenosine triphosphatase complex from the wild-type and N,N'-dicyclohexylcarbodiimide-resistant strains of Streptococcus faecalis.

We have purified the F1-F0 adenosine triphosphatase complex from wild-type Streptococcus faecalis ATCC 9790 and an N,N'-dicyclohexylcarbodiimide (DCCD)-resistant mutant strain, SF-dcc-8. For preliminary purification of the complex, reconstituted F1-F0, prepared from isolated F1 adenosine triphosphatase and depleted membranes, was extracted with sodium deoxycholate and fractionated by salt precipitation. By means of two-dimensional gel electrophoresis, the F1-F0 complex was purified as a single, catalytically active band in the first dimension and then resolved into constituent subunits under denaturing conditions in the second dimension. The electrophoretic purification of F1-F0 removed a delta-less form of F1 as well as other impurities, including lipoteichoic acid. Both the DCCD-sensitive and the DCCD-resistant F1-F0 adenosine triphosphatase appeared to consist of eight proteins, five of which corresponded to the F1 subunits alpha, beta,, gamma, delta, and epsilon. The F0 sector proteins, designated M27, M15, and M6, had Mr values of 27,000, 15,000, and 6,000, respectively. There appear to be multiple copies of M6 in the complex. [14C]DCCD reacted specifically and covalently with M6 in the wild-type F1-F0 but failed to label the M6 protein in the complex from the DCCD-resistant strain. It is suggested that DCCD resistance in the SF-dcc-8 mutant may be due to a modification of the M6 protein which hinders access of DCCD to the reactive site.