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L D Schultz

Publications and source records attributed to L D Schultz.

At least 37 records · Page 2Linked to original sources
PubMed1990

Purification and characterization of human plasminogen activator inhibitor type I expressed in Saccharomyces cerevisiae.

The rapidly acting inhibitor of plasminogen activators, PAI-1, was produced intracellularly in Saccharomyces cerevisiae by using the ADH2 promoter to drive the expression of the human PAI-1 cDNA. Approximately 8 mg of human PAI-1 was produced per liter of confluent yeast culture. A purification scheme which resulted in 20% recovery of isolated PAI-1 from the broken yeast cell homogenate was devised. Yeast-derived human PAI-1 differs from endothelial-type PAI-1 isolated from HT1080 fibrosarcoma cells in that the recombinant inhibitor does not contain carbohydrate side chains. Nevertheless, the activity and other functional attributes of yeast-derived PAI-1 are similar to those exhibited by HT1080 fibrosarcoma cell-derived PAI-1. Hence, this study demonstrates that expression of human PAI-1 in yeast is a viable strategy for the production of ample quantities of this key modulator of plasminogen activator-mediated proteolysis.

Antigens↗
PubMed1990

Yeast-expressed p55 precursor core protein of human immunodeficiency virus type 1 does not elicit protective immunity in chimpanzees.

Yeast-expressed p55 precursor core protein of human immunodeficiency virus type 1 (HIV-1) was used to immunize chimpanzees. The animals developed high titers of antibodies to p55 as well as to the p24 and p17 mature cleavage products of the core precursor. Virus-neutralizing antibodies were not elicited. The induced immune responses did not prevent establishment of HIV-1 infection following challenge of one immunized chimpanzee with live virus.

Animals↗
PubMed1989

Expression and secretion of biologically active echistatin in Saccharomyces cerevisiae.

A synthetic gene coding for a platelet aggregation inhibitor, echistatin (ECS), was inserted into a Saccharomyces cerevisiae expression vector utilizing the alpha-mating factor pre-pro leader sequence and galactose-inducible promoter, GAL10. Cleavage of the pre-pro leader sequence in vivo results in the secretion of a properly processed recombinant ECS with the native N-terminal glutamic acid residue. Recombinant ECS was recovered from yeast supernatants and purified by reverse phase high performance liquid chromatography. Recombinant ECS expressed and purified from yeast was identical to native ECS in its ability to inhibit platelet aggregation.

Amino Acid Sequence↗
PubMed1989

Purification and characterization of human immunodeficiency virus (HIV) core precursor (p55) expressed in Saccharomyces cerevisiae.

The core structure of retroviruses, including the human immunodeficiency virus (HIV), consists of proteins that are initially synthesized as polyprotein precursors and then processed by a virally encoded protease yielding the mature core polypeptides. To obtain sufficient quantities of the purified HIV core precursor p55 for detailed studies, a segment of HIV DNA encoding the full length core precursor polyprotein p55 was expressed in Saccharomyces cerevisiae using a plasmid containing a constitutive galactose promoter. The expression of this DNA produced a protein with an estimated molecular size of 55,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); this protein was immunoreactive to anti-HIV p24 antisera. Following cell lysis, freezing, and thawing, the expressed protein was an insoluble aggregate that served as the starting material for the purification process. Solubilization of the insoluble p55 with guanidine HCl followed by phenyl-Sepharose column chromatography and high performance liquid chromatography resulted in a preparation of p55 that was greater than 95% pure by SDS-PAGE, immunoreactive to anti-HIV core protein antibodies, and completely soluble in aqueous solution. The expressed p55 appeared to be myristoylated as evidenced by the incorporation of radiolabel following incubation of recombinant yeast cells with [3H]myristic acid; in addition the amino terminus of the final purified protein was blocked. Proteolytic digestion of purified p55 with synthetic HIV protease yielded the predicted amino- and carboxyl-terminal products; these were confirmed by amino acid sequence analysis. In contrast, digestion of purified p55 by the protease derived from the avian myeloblastosis virus resulted in fragments that were different in size from those produced by the HIV protease. The availability of the purified, full length water-soluble HIV core precursor will be useful in identifying agents that inhibit its processing by the HIV protease.

Amino Acid Sequence↗
PubMed1989

The application of molecular biology to the development of novel vaccines.

In summary, we have shown that yeast is the preferred host for the expression of recombinant-derived hepatitis B vaccines, and that a yeast expression system which is productive, stable and scaleable can be developed for each of the three HBV envelope proteins. The versatility of regulated and integrated yeast expression systems in the production of foreign polypeptides with biomedical utility also has been highlighted. We also have shown that careful attention to the development of recombinant clones helps to optimize the entire production process leading to highly purified products which share many biochemical properties with the plasma-derived vaccine. Furthermore, immunization with PreS2 sequences is capable of protecting chimpanzees from HBV infection. The availability of PreS2 + S and PreS1 + PreS2 + S proteins expressed in yeast now provides the opportunity for establishing the relevance of such candidate vaccines in preventing human disease, thereby highlighting the utility of molecular biology in modern vaccine development.

Animals↗
PubMed1988

Antigenic analysis of the Epstein-Barr virus major membrane antigen (gp350/220) expressed in yeast and mammalian cells: implications for the development of a subunit vaccine.

The Epstein-Barr virus (EBV) major surface membrane antigen, gp350/220, was expressed in recombinant yeast cells and in several recombinant mammalian cell lines. Each of the expressed proteins was analyzed for its ability to bind to a panel of anti-gp350/220 monoclonal antibodies and to a series of anti-EBV positive human sera. The antigens also were used as immunogens for the immunization of rabbits. Each expressed protein was found to be unique both in its pattern of reactivity to the various antibodies and in the spectrum of antibody induced following animal immunization. These results suggest that cell-specific post-translational modifications critically influence the antigenic presentation of the expressed proteins. Nonetheless, all of the mammalian cell-derived versions of the membrane antigen were found capable of inducing EBV-specific neutralizing antibodies.

Animals↗
PubMed1987

Regulated overproduction of the GAL4 gene product greatly increases expression from galactose-inducible promoters on multi-copy expression vectors in yeast.

High-level, galactose-inducible expression originating from GAL promoters in Saccharomyces cerevisiae is mediated by highly specific interactions between the GAL4-coded protein and nucleotide sequences. The potential utility of recombinant GAL promoter/foreign gene constructions for the regulatable and high-level expression of foreign proteins in yeast is well recognized. However, the utility of this system is limited severely in the case of multiple copies of such constructions due to the very low level of the GAL4-coded protein and to the loss of inducibility which occurs if levels of the GAL4 protein are amplified constitutively. To surmount these limitations, we have constructed a novel yeast strain which overproduces the GAL4 protein in a regulated fashion. This 'integrant' strain contains an integrated copy of a hybrid gene consisting of the galactose-inducible GAL10 promoter fused to the GAL4 structural gene. In the absence of galactose, the integrant strain and the isogenic 'non-integrant' parental strain show only a basal level of transcription from the constitutively active chromosomal GAL4 gene. However, following the addition of galactose to the culture medium, the 'integrant' strain synthesizes at least 20-fold more GAL4 mRNA and substantially more GAL4 protein than the 'non-integrant' strain. A high-copy-number expression vector containing the GAL10 promoter and alpha mating factor pre-pro leader fused to the structural gene for Epstein-Barr virus gp350 was introduced into both types of cells. The resulting transformed 'integrant' cells produced approximately five-fold more gp350 mRNA and ten-fold more gp350-related proteins than the transformed 'non-integrant' cells following galactose induction. This 'integrant' strain should prove generally useful for the maximal, regulated expression in yeast of structural genes driven by a galactose-inducible promoter.

DNA, Recombinant↗
PubMed1987

Expression and secretion in yeast of a 400-kDa envelope glycoprotein derived from Epstein-Barr virus.

The major envelope glycoprotein (gp350) of Epstein-Barr virus has been expressed and secreted in the yeast Saccharomyces cerevisiae as a 400-kDa glycoprotein. This is the first example of the secretion of such a large, heavily glycosylated heterologous protein in yeast. Since gp350 proved highly toxic to S. cerevisiae, initial cellular growth required repression of the expression of gp350. Using temperature- or galactose-inducible promoters, cells could be grown and the expression of gp350 then induced. After induction, the glycoprotein accumulated both intracellularly as well as in the culture medium. Only the most heavily glycosylated form was secreted, suggesting a role for N-linked glycans in directing secretion. The extent of O-linked glycosylation of the yeast-derived protein was similar to that of the mature viral gp350. N-linked glycosylation varied slightly depending upon culture conditions and host strain used and was more extensive than that associated with the mature viral gp350. Although there is no evidence that more than a single mRNA for the glycoprotein was expressed from the recombinant plasmid, variously sized glycoproteins accumulated in yeast at early stages after induction, probably reflecting intermediates in glycosylation. The yeast-derived glycoproteins reacted with animal and human polyclonal antibodies to gp350 as well as with a neutralizing murine monoclonal antibody to gp350, suggesting that this glycoprotein retains several epitopes of the native glycoprotein.

Cloning, Molecular↗
PubMed1986

The safety of the hepatitis B vaccine. Inactivation of the AIDS virus during routine vaccine manufacture.

In the United States, one hepatitis B vaccine (Heptavax-B) has been licensed for the prevention of hepatitis B virus infections. Even though this vaccine has been shown to be highly effective and well tolerated in controlled trials and has been recommended for use in those at risk for acquiring infection by hepatitis B virus, many individuals have been reluctant to be immunized for fear of contracting acquired immunodeficiency syndrome (AIDS). In this study, we demonstrate that each of the three inactivation steps used in the manufacture of Heptavax-B independently will inactivate the infectivity of high-titered preparations of the AIDS virus; recipients of the hepatitis B vaccine do not develop antibodies to the AIDS virus; the hepatitis B vaccine does not contain detectable levels of nucleic acids related to the AIDS virus. These observations clearly demonstrate that vaccination with the currently available hepatitis B vaccine poses no demonstrable risk for acquiring AIDS.

Antibodies, Viral↗
PubMed1983

Nucleotide sequence of the tcml gene (ribosomal protein L3) of Saccharomyces cerevisiae.

The yeast tcml gene, which codes for ribosomal protein L3, has been isolated by using recombinant DNA and genetic complementation. The DNA fragment carrying this gene has been subcloned and we have determined its DNA sequence. The 20 amino acid residues at the amino terminus as inferred from the nucleotide sequence agreed exactly with the amino acid sequence data. The amino acid composition of the encoded protein agreed with that determined for purified ribosomal protein L3. Codon usage in the tcml gene was strongly biased in the direction found for several other abundant Saccharomyces cerevisiae proteins. The tcml gene has no introns, which appears to be atypical of ribosomal protein structural genes.

Amino Acid Sequence↗
PubMed1981

In vitro RNA synthesis in oocyte nuclei of the newt Notophthalmus.

An incubation medium is described which supports RNA synthesis in isolated oocyte nuclei of the newt Notophthalmus, and which permits subsequent autoradiographic examination of the lampbrush chromosomes and nucleoli. By using different concentrations of alpha-amanitin we distinguish RNA synthesis due to RNA polymerases I, II and III. All RNA synthesis on loops is inhibited by 0.5 microgram/ml of alpha-amanitin and is therefore due to polymerase II. Polymerase III is responsible for RNA synthesis at a small number of discrete sites in condensed chromatin. These include the centromere bars of three of four chromosomes, which probably represent 5S RNA synthesis, as well as 15-20 lesser sites scattered elsewhere. Polymerase I activity is confined to the nucleoli.

Amanitins↗
PubMed1980

Processing of intervening sequences: a new yeast mutant which fails to excise intervening sequences from precursor tRNAs.

By using conditional loss of suppression an an assay, we have been successful in screening for a yeast mutant which is defective in tRNA processing. The los1-1 mutation causes an accumulation of a subset of precursor tRNAs at the nonpermissive temperature. These pre-tRNAs are like those which accumulate in the yeast mutant ts 136 (rna1) in that they have transcribed intervening sequences. The mutations at los1-1 and rna1 complement and segregate independently of each other. The los1-1 mutation affects the expression of all 8 tyrosine-inserting suppressor loci, but does not seem to affect rRNA or mRNA synthesis.

Base Sequence↗
PubMed1977

Occurrence and distribution of interdental gingival clefts following orthodontic movement into bicuspid extraction sites.

Forty patients in active retention following orthodontic tooth movement into premolar extraction sites were examined for the occurrence and distribution of interdental gingival clefts, defined as an invagination of interproximal tissue with definite mesial and distal peaks having a depth of at least 1 mm. Fourteen of the forty orthodontic patients demonstrated clefts in one or more of the premolar extraction sites. No clefts were observed in premolar areas of orthodontic patients who did not require premolar extraction or in patients without previous orthodontic treatment. Interdental clefts occurred most frequently at the buccal aspect of mandibular first premolar extraction sites. The presence of the cleft appears to have clinical implications, both in terms of orthodontic relapse, and maintenance of gingival health.

Adolescent↗
PubMed1976

Transcription in yeast: alpha-amanitin sensitivity and other properties which distinguish between RNA polymerases I and III.

Three peaks of DNA-dependent RNA polymerase (RNA nucleotidyltransferase) activity are resolved by chromatography of a sonicated yeast cell extract on DEAE-Sephadex. The enzymes, which are named RNA polymerases I, II, and III in order of elution, show similar catalytic properties to the vertebrate class I, class II, and class III RNA polymerases, respectively. Yeast RNA polymerase III is readily distinguished from yeast polymerase I by its biphasic amnonium sulfate activation profile with native DNA templates, greater enzymatic activity with poly[d(I-C)] than with native salmon sperm DNA, and distinctive chromatographic elution positions from DEAE-cellulose (0.12 M ammonium sulfate) compared with DEAE-Sephadex (0.32 M ammonium sulfate). The three yeast RNA polymerases also show significant differences in alpha-amanitin inhibition. RNA polymerase II is the most sensitive (50% inhibition at 1.0 mug of alpha-amanitin per ml). Contrary to the results for vertebrate systems, yeast polymerase I can be completely inhibited by alpha-amanitin at high concentrations (50% inhibition at 600 mug/ml) while yeast RNA polymerase II BEGINS TO SHOW SIGNIFICANT INHIBITION ONLY AT CONCENTRATIONS EXCEEDING 1 MG/ML. Therefore, yeast RNA polymerases I and III show a pattern of alpha-amanitin sensitivity that is the reverse of that seen for the analogous vertebrate RNA polymerases.

Amanitins↗
PubMed1972

Transcription in yeast: separation and properties of multiple FNA polymerases.

Four peaks of DNA-directed RNA polymerase activity are resolved by salt gradient elution of a sonicated yeast cell extract on DEAE-Sephadex. The enzymes, which are named IA, IB, II, and III in order of elution, all appear to come from cell nuclei. Only enzyme II is sensitive to alpha-amanitin. All enzymes are more active with Mn(++) than with Mg(++) as divalent ion. Enzymes IB and II have salt optima in the range 0.05-0.10 M (NH(4))(2)SO(4), whereas enzyme III is maximally active at 0.20-0.25 M (NH(4))(2)SO(4). With optimal salt concentration and saturating DNA, the template preference ratio, activity on native calfthymus DNA divided by activity on denatured calf-thymus DNA, is 2.2 for IB, 0.4 for II, and 3.5 for III. None of the yeast polymerases was inhibited by rifamycin SV. Rifamycin AF/013 effectively inhibited polymerases IB, II, and III.

Ammonium Sulfate↗