Size-exclusion High-pressure Liquid Research Articles

Expression and secretion of an assembled tetrameric CH2-deleted antibody in E. coli

We have expressed in E. coli a functional assembled antibody variant that is secreted into the media. The antibody variant is a CH2-deleted chimeric antibody 14.18, which was previously shown to be a potentially useful reagent for radioimmunodetection of human tumors. The bacterial expression vector contains a dicistronic unit comprised of a L-chain cDNA and a CH2-deleted H-chain cDNA. For translocation across the bacterial membranes, we have replaced the natural signal peptides of the H and L chains with the signal peptide of the bacterial protein pectate lyase B. When expressed in the JM105 host under the control of the trp-lac promoter, the products were secreted into the M9 growth media to about 350 micrograms/L. The secreted antibody, which can be readily purified from the media without any denaturation or renaturation steps, retains antigen-binding activity. SDS-PAGE and nondenaturing size exclusion high-pressure liquid chromatography showed that it is a mixture of assembled HL and fully assembled H2L2. In H2L2, inter-H chain disulfide bonds are not formed, and the two HL half-molecules are likely held together by the trans interaction between the two CH3 domains.

Effects of recombinant insulin-like growth factor-I (IGF-I) and growth hormone on serum IGF-binding proteins in calorically restricted adults.

To determine the effects of exogenous insulin-like growth factor-I (IGF-I) and GH on IGF-binding proteins (IGFBP)-1, -2, and -3, six healthy nonobese adult volunteers underwent two 2-week periods of diet restriction (20 Cal/kg.day), and during the last 6 days of the first period received either IGF-I (12 micrograms/kg.h by iv infusion over 16 h) or GH (0.05 mg/kg.day by sc injection). During the second 2-week study period, the alternate hormone was given. IGFBP-1 and -2 concentrations were determined by specific RIA, and changes in IGFBP-3 were assessed by ligand blotting. Free IGF-I concentrations were measured by size-exclusion high pressure liquid chromatography, followed by RIA. Diet restriction alone did not affect either IGFBP-1 or -2 significantly. IGF-I treatment increased IGFBP-1 from 78 +/- 46 ng/mL (mean pretreatment) to 137 +/- 64 ng/mL (P less than 0.001; mean for the last 4 days of IGF-I). IGF-I also caused an increase in IGFBP-2 from 315 +/- 136 to 675 +/- 304 ng/mL (P less than 0.001). GH injections caused a modest decline in IGFBP-1 concentrations but had no effect on IGFBP-2 concentrations. By ligand blotting, both IGF-I and GH caused a modest increase in IGFBP-3 band intensity. In three subjects diet restriction alone caused a small decrease in IGFBP-3 hand intensity, and this was reversed by hormone treatment. Free IGF-I concentrations in serum were increased from 1.6% to 4.4% of the total IGF-I during IGF-I infusions. GH injections caused a smaller increase in free IGF-I concentrations. The results show significant increases in IGFBP-1 and -2 during IGF-I infusion. The change in IGFBP-3, while significant, is quantitatively less than that in experimental animals that have been given IGF-I while undergoing dietary restriction. The net effect of the changes in these three forms of IGFBPs is not sufficient to maintain a normal IGF-I-binding capacity in serum, because free IGF-I levels were increased disproportionately during the IGF-I infusions. Because hypoglycemia was noted in these subjects despite insulin suppression, these alterations in IGFBPs might have changed the tissue bioavailability of IGF-I and facilitated its hypoglycemic effects.

Acetate-dependent methylation of two corrinoid proteins in extracts of Methanosarcina barkeri

Corrinoid proteins have been implicated as methyl carriers in methane formation from acetate, yet specific corrinoid proteins methylated by acetate-derived intermediates have not been identified. In the presence of ATP, H2, and bromoethanesulfonic acid, label from 3H- or 2-14C-labeled acetate was incorporated into the protein fraction of cell extracts of Methanosarcina barkeri. Incorporated label was susceptible to photolysis, yielding labeled methane as the anaerobic photolysis product. Size exclusion high-pressure liquid chromatography (HPLC) demonstrated the presence of at least three labeled proteins with native molecular sizes of 480, 200, and 29 kDa, while electrophoresis indicated that four major labeled proteins were present. Dual-label experiments demonstrated that these four proteins were methylated rather than acetylated. Two of the proteins (480 and 29 kDa) contained the majority of radiolabel and were stably methylated. After labeling with [2-14C]acetate, the stable 14CH3-proteins were partially purified, and 14CH3-cofactors were isolated from each protein. UV-visible spectroscopy and HPLC demonstrated these to be methylated corrinoids. When the 480-kDa corrinoid protein was purified to 70% homogeneity, the preparation was found to have subunits of 40 and 30 kDa. The 480-kDa protein but not the 29-kDa protein was methylated during in vitro methanogenesis from acetate and demethylated as methanogenesis ceased, consistent with the involvement of this protein in methane formation.

Functional liver imaging with 99 Tcm-galactosyl-neoglycoalbumin (NGA) in alcoholic liver cirrhosis and liver fibrosis

Synthesis of 99Tcm-galactosyl-neoglycoalbumin (99Tcm-NGA), a recently described new radioligand with high specificity for hepatic binding protein (HBP), a galactose-residue specific receptor on hepatocytes, was carried out by covalent coupling of 2-imino-2-methoxyethyl-1-thio-beta-D-galactopyranoside to the primary amino groups of human serum albumin. NGA was purified by ultrafiltration and size exclusion high-pressure liquid chromatography (HPLC). Using a computer-based simulation program, time-activity data for the liver and precordium, blood radioactivity 2 min after i.v. injection of the radioligand, the association constant of 99Tcm-NGA-binding to HBP measured on human liver biopsies in vitro, and other patient-related parameters were put into a five-state non-linear model describing the pharmacokinetics of 99Tcm-NGA. By fitting the parameters of the model iteratively to the experimental data, an estimate of HBP concentration in the liver and of total liver blood flow was obtained. Using this model we studied 32 patients (53 +/- 11 years) with different clinical stages of alcoholic liver cirrhosis. Child B and Child C stage cirrhotic patients had a lower HBP concentration in the liver compared to control individuals (0.96 +/- 0.21 mumol l-1). The group with the most advanced cirrhosis (Child C stage) had a significantly lower HBP concentration (0.27 +/- 0.15 mumol l-1) than Child A patients (0.66 +/- 0.21 mumol l-1; P less than 0.01) and Child B patients (0.53 +/- 0.18 mumol l-1; P less than 0.05). In four patients with biopsy-proven liver fibrosis (0.84 +/- 0.20 mumol l-1) no difference in receptor concentration from normal individuals was estimated. Changes in liver blood flow were not significant between the groups. A direct comparison of HBP concentration estimated in vivo by 99Tcm-NGA functional imaging and HBP concentration measured in vitro on a surgically removed liver biopsy specimen from the same patient with a normal liver and liver cirrhosis showed good matching of these two values. The results suggest that in vivo estimation of HBP concentration in the liver by 99Tcm-NGA functional imaging might be a suitable method for determining liver cell mass.

Scrapie‐associated precursor proteins

We describe the antigenic properties and detection of a normal isoform of scrapie-associated precursor protein (PrP33-35C) in normal, and both normal and scrapie isoforms in scrapie- or Creutzfeldt-Jakob disease (CJD)-infected mouse, hamster, and human brains, using a variety of specific antibodies. Polyclonal antibodies raised against mouse and hamster PrP27-30 and against a synthetic peptide of the N-terminal sequence of this protein were used as immunologic probes. PrP27-30 purified as a primary immunogen corresponded to the lower molecular mass peptide, with Mr between 9.3 and 13.5 kd as estimated by size-exclusion high-pressure liquid chromatography. ELISA and immunoblot techniques demonstrated that antibodies recognized homologous antigens as well as precursor proteins from brains (PrP33-35C) and the scrapie isoform of scrapie-associated proteins (PrP33-35Sc/CJD and PrP27-30) from scrapie- and CJD-infected brains. The normal, scrapie, and CJD isoforms of scrapie-associated proteins share common epitopes with varying degrees of interspecies homology. Specific antigen detected in neurons indicated that these proteins are synthesized primarily in these cells. In infected brains, extracellular amyloid deposits formed by the scrapie isoform of PrP protein also strongly reacted with anti-PrP antibodies.

A direct microassay for serum retinol (vitamin A alcohol) by using size-exclusion high-pressure liquid chromatography with fluorescence detection

Serum retinol (bound to plasma retinol-binding protein, RBP) can be determined by direct injection of as little as 20 μl of serum or plasma by using size-exclusion high-pressure liquid chromatography (SE-HPLC) with fluorescence detection. Toyo Soda TSK G-3000SW columns (0.75 × 7.5-cm guard column plus 0.75 × 30-cm analytical column) were eluted with 0.2 m NaCl/0.01 m phosphate buffer (pH 6.8) at 1 ml/min, with detection at 280 nm for protein elution. Fluorescence of the retinol-RBP complex was monitored with excitation at 334 nm (interference filter) and emission at 425 nm (long-pass filter). The retinol-RBP complex eluted as two peaks, the holo-RBP-transthyretin complex (apparent molecular weight 70,000) and holo-RBP (apparent molecular weight 9000). Identities of these peaks were established by immunodiffusion assay of the proteins and by extraction and analysis of retinol. Nonideal interactions with the column packing seem to be responsible for the low apparent molecular weight of holo-RBP. The first peak predominated when large volumes of serum (100 to 250 μl) were injected, and the second when small volumes (5 to 50 μl) were analyzed. The integrated area of the two fluorescence peaks due to retinol bound to RBP was proportional to the volume of a serum sample injected over the range 5 to 250 μl. Rat serum samples were analyzed for retinol by SE-HPLC and by conventional extraction and HPLC analysis; over the range 4 to 67 μg retinol/dl serum (0.14 to 2.34 μ m), a linear relationship was observed between the results of the two assay techniques ( n = 25, r = 0.93). Reproducibility was ±4% within-day, ±6% between days. At a signal:noise ratio of 5:1, the minimum amount of retinol quantitable was 1 ng, corresponding to 5 μg retinol/dl (0.17 μ m) for a 20-μl injection.

Isolation of a glucan-binding domain of glucosyltransferase (1,6-alpha-glucan synthase) from Streptococcus sobrinus.

A glucan-binding domain of 1,6-alpha-glucan synthase (dextransucrase) (GTF-S) was isolated from a trypsin digest of the Streptococcus sobrinus enzyme. The large 60.5-kilodalton peptide had an affinity for dextran comparable to that of the native enzyme, but had no glucan synthesis activity. The domain was produced in high yield compared with other large cleavage products, which allowed easy purification by size exclusion high-pressure liquid chromatography and affinity chromatography. Two other proteases (mouse submaxillaris protease and lysyl endopeptidase) with specificities similar to trypsin generated a distribution of GTF-S peptides that was also greatly enriched in the glucan-binding peptide. Proteases with markedly different specificities (chymotrypsin and Staphylococcus aureus V8 protease) produced a family of peptides with some evidence of the glucan-binding domain but in far lower yield. The tertiary structure of the domain was critical to its resistance to proteolysis; heat denaturation of GTF-S before trypsin digestion resulted in cleavage of the enzyme to small limit peptides leaving no evidence of the glucan-binding domain. The amino acid composition of the peptide was very similar to that of the native enzyme. The common occurrence of proteases in oral streptococcus cultures and reports of glucosyltransferase degradation during purification and storage raises the possibility that some accounts of glucan-binding receptors are peptides derived from glucosyltransferase. Kinetic implications of a glucan-binding domain are discussed.

Characterization of circulating Yersinia-specific immune complexes in patients with yersiniosis

The size of immune complexes (ICs) containing Yersinia enterocolitica antigens was studied by size exclusion high-pressure liquid chromatography and sucrose density gradient ultracentrifugation in sera of patients with recent yersiniosis. The ICs detected were relatively small, i.e., of equal size to or slightly larger than the corresponding anti-Yersinia antibodies. The size of the ICs was equal in the patients with Yersinia-triggered reactive arthritis and in those recovering without complications. No changes were observed during a follow-up. The equal size of ICs in the patients with and without arthritis also suggests that antigens and antibodies involved are similar in both patient groups. Taken together with our earlier findings indicating occurrence of high concentrations of Yersinia IgM ICs in the arthritic patients, the present results suggest that Yersinia-IgM ICs have a role in the pathogenesis of Yersinia-triggered reactive arthritis.

Purification and characterization of a polypeptide from chick brain that promotes the accumulation of acetylcholine receptors in chick myotubes.

Acetylcholine receptors (AChRs) are packed in the postsynaptic membrane at neuromuscular junctions at a density of approximately 20,000/micron 2, whereas the density a few micrometers away is less than 20/micron 2. To understand how this remarkable distribution comes about during nerve-muscle synapse formation, we have attempted to isolate factors from neural tissue that can promote the accumulation of AChRs and/or alter their distribution. In this paper we report the purification of a polypeptide from chick brains that can increase the rate of insertion of AChR into membranes of cultured chick myotubes at a concentration of less than 0.5 ng/ml. Based on SDS PAGE and the action of neuraminidase, the acetylcholine receptor-inducing activity (ARIA) appears to be a 42,000-D glycoprotein. ARIA was extracted in a trifluoroacetic acid-containing cocktail and purified to homogeneity by reverse-phase, ion exchange, and size exclusion high pressure liquid chromatography. Dose response curves indicate that the activity has been purified 60,000-fold compared with the starting acid extract and approximately 1,500,000-fold compared with a saline extract prepared from the same batch of brains. Although the ARIA was purified on the basis of its ability to increase receptor incorporation, we found that it increased the number and size of receptor clusters as well. It is not yet clear if the two effects are independent. The 42-kD ARIA is extremely stable: it was not destroyed by exposure to intact myotubes, low pH, organic solvents, or SDS. Its action appears to be selective in that the increase in the rate of receptor insertion was not accompanied by an increase in the rate of protein synthesis. Moreover, there was no change in cellular, surface membrane, or secreted acetylcholinesterase. The effect of ARIA is apparently independent of the state of activity of the target myotubes as its effect on receptor incorporation added to that of maximal concentrations of tetrodotoxin.

Isolation and characterization of the heat stable enterotoxin from a pathogenic bovine strain of Escherichia coli

A heat-stable enterotoxin secreted by a pathogenic strain of Escherichia coli of calf origin was purified to homogeneity by a procedure involving acetone fractionation, DEAE cellulose chromatography, Biogel P 2 chromatography and size exclusion high pressure liquid chromatography. The purity of the product was ascertained by amino-acid analyses and amino acid sequence using manual degradation with 4-N, N dimethylaminoazobenzene-4′ isothiocyanate (DABITC) and an automatic gas phase sequenator. The following amino acid sequence is proposed : Asn-Thr-Phe-Tyr-Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys-Tyr-. It is identical to a similar active peptide isolated from strains of porcine origin. Antibodies to ST were successfully produced in rabbits using a conjugate with bovine serum albumin. The ultraviolet absorption and circular dichroism spectra of the active product were recorded and discussed.

Tropomyosin-like properties of clathrin light chains allow a rapid, high-yield purification.

The light chains (LCa and LCb) of bovine brain clathrin are resistant to heat denaturation by boiling, a property shared by tropomyosin (Bailey, K., 1948, Biochem. J., 43:271-281). Light chains were partially purified by boiling and centrifugation of a Tris-extract of crude membranes prepared from bovine brains (Keen, J. H., M. C. Willingham, and I. H. Pastan, 1979, Cell., 16:303-312). Contaminant polypeptides were then removed by size-exclusion high-pressure liquid chromatography. The purified light chains were separated from each other by using an immunoaffinity column prepared from a monoclonal antibody CVC.7 specific for LCa and not LCb.

Antigenic determinants of the HLA-B7 molecule; Bw6- and B7-specific determinants are spatially separate.

Monoclonal antibodies that bind HLA-B7 were used to show that the B7-specific determinant is at a topologically different site from that of the broad polymorphic, Bw6 determinant. The relationship to other antigenic determinants defined by monoclonal antibodies was also assessed. These results were independently obtained in four ways: (1) by cellular blocking assays, in which there was no inhibition of 125I-B7 antibody binding in the presence of Bw6 antibody and no inhibition of 125I-Bw6 antibody binding in the presence of B7 antibody; (2) cellular binding assays under conditions of antibody saturation showed the binding of B7-specific and Bw6 antibodies were additive; (3) solid-phase radioimmune assays demonstrated enhancement between B7-specific and Bw6 antibodies; (4) analysis of antigen antibody complexes by size-exclusion high pressure liquid chromatography showed Bw6 and B7 antibodies could form tetramolecular complexes with papain-solubilized HLA-B7. Limitations were encountered in using cellular blocking assays to map antigenic determinants of HLA-B7. These assays can produce blocking in cases where two antibodies are not competing for an antigenic determinant. Mapping antigenic determinants with assays using purified HLA-B7 as the antigenic target, in addition to cell-based assays, provided a more accurate picture.