Biomedical subjects

A Shatzman

Publications and source records attributed to A Shatzman.

9 recordsLinked to original sources

PubMed1992

Effector-assisted refolding of recombinant tissue-plasminogen activator produced in Escherichia coli.

Recombinant tissue-plasminogen activator (r-tPA), expressed in Escherichia coli cells in an aggregated form, was solubilized with a strong chaotrope in the absence of any reducing agent. The solubilized molecule was reactivated by a procedure that was developed to mimic the physiological conditions optimal for the functional folding and activity of the native protein. The use of partially purified fibrinogen, as a source of fibrin (the effector), is shown to facilitate the reactivation process and increase its yield by at least a factor of two. The yield of the process is also shown to be particularly dependent on the recombinant protein concentration. At a concentration level of 3-3.7 mg r-tPA/L in the reactivation mixture, up to a 90% yield of activity was obtained. Purification of the activated form of r-tPA was achieved with a two-step column-chromatography scheme. This included a gel filtration step on a Sephadex G-50 column followed by an affinity chromatography step on a lysine-sepharose column. The product was composed of roughly equal amounts of one-chain and two-chain t-PA. The feasibility of using a two water-soluble polymeric phase system, with a centrifugal partition chromatography (CPC), in scaling up the reactivation process or the purification step was also evaluated.

Chromatography, Affinity↗

PubMed1990

The genetic analysis of the HIV envelope binding domain on CD4.

Through mutagenesis, we identified a single high-affinity binding site for gp120 on the human CD4 protein. This site is localized in the V1 domain within residues 41 to 55. The collection of mutants was also used to define the epitopes for 55 anti-CD4 monoclonal antibodies. The locations of these epitopes are consistent with a V kappa-like structure for the V1 domain. In the context of this structure, the gp120 binding site encompasses the small CDR2 loop. Through deletion mutagenesis at the termini of the V1 domain, we further defined the minimal region required to retain high-affinity binding to gp120. Short deletions at both termini disrupt binding to gp120 and recognition by conformation-sensitive anti-CD4 monoclonal antibodies. We conclude that amino acids at both the amino and carboxy termini are critical to the conformation of the V1 domain and, in particular, to the integrity of the gp120 binding site.

Antibodies, Monoclonal↗

PubMed1989

Increasing gene expression in yeast by fusion to ubiquitin.

Heterologous gene expression in yeast can be increased up to several hundred-fold by expressing a foreign gene as a fusion to the ubiquitin gene. An endogenous yeast endoprotease (Ub-Xase) removes the ubiquitin from the fusion product to produce the authentic protein. The utility of this technique has been demonstrated by expression of three different proteins in yeast as both unfused and ubiquitin-fused forms: 1) the alpha subunit of the mammalian stimulating G-protein of the adenylate cyclase complex (Gs alpha); 2) a soluble fragment of the T cell receptor protein (sCD4); and 3) the protease domain of human urokinase (UKP). The sequence specificity of the Ub-Xase was demonstrated by mutagenesis of the carboxyl-terminal glycine of ubiquitin to an alanine, which inhibited ubiquitin removal in vivo. Processing of the ubiquitin-Gs alpha fusion protein (ub-Gs alpha) in vivo resulted in Gs alpha which could be reconstituted in mammalian membrane preparations and had the same specific activity as the authentic Gs alpha expressed in yeast. The yeast Ub-Xase has also been shown to work in vitro by the processing of a ub-sCD4 fusion protein synthesized in Escherichia coli. This technology should greatly enhance the utility of yeast for heterologous protein production.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1989

Ubiquitin function studied by disulfide engineering.

Disulfide engineering was used to probe the role of conformational mobility in ubiquitin-mediated proteolysis. Six genes that encode cysteine-containing mutants of ubiquitin were constructed, expressed in Escherichia coli and the proteins purified. Single cysteine-containing mutants and a 4/14 disulfide were active in degradation of a substrate protein in vitro, while the 4/66 disulfide, which cross-links the NH2- and COOH-terminal strands of the protein, was only 20-30% active. The solution structure of the 4/66 mutant was solved: the disulfide is left-handed with no perturbations in the backbone from that of wild type ubiquitin. The results suggest that conformational mobility is required for the activity of ubiquitin in signaling proteolysis.

Computer Simulation↗

PubMed1988

Efficient expression of the yeast metallothionein gene in Escherichia coli.

The yeast metallothionein gene CUP1 was cloned into a bacterial expression system to achieve efficient, controlled expression of the stable, unprocessed protein product. The Escherichia coli-synthesized yeast metallothionein bound copper, cadmium, and zinc, indicating that the protein was functional. Furthermore, E. coli cells expressing CUP1 acquired a new, inducible ability to selectively sequester heavy metal ions from the growth medium.

Amino Acid Sequence↗

PubMed1987

High-level production of fully active human alpha 1-antitrypsin in Escherichia coli.

The human alpha-1-antitrypsin (A1AT) gene expressed in Escherichia coli as a full-length, non-fusion gene product accumulates to a relatively low level approaching less than or equal to 0.1% of total cellular protein. In contrast, deletion of the first 5, 10 or 15 codons leads to production of truncated A1AT derivatives at levels between 10 and 30% of total cellular protein. The protein with the largest truncation was insoluble and inactive following solubilization by chaotropic agents. In contrast, the two derivatives with the smaller truncations were found to be soluble, and exhibit identical specific activities in both trypsin and elastase inhibition assays to authentic human A1AT. The expression of the full-length A1AT was also optimized by making silent third position mutations within its first 15 codons. These mutations were chosen to optimize codon usage and minimize the possibility of RNA secondary structure formation in this region. Via this approach, expression of full-length, authentic, fully active A1AT was increased at least 20-fold to 2% of total cellular protein. Optimal expression was obtained using as few as three silent mutations in the first five codons, confirming the importance of this 5'-terminal region as had been defined by our deletion mutants. Both the full-length derivatives as well as the small N-terminal deletion derivative can be readily purified from bacterial extracts in fully active form suitable for the examination of their potential therapeutic application.

Base Sequence↗

PubMed1987

Selection of mutations that increase alpha 1-antitrypsin gene expression in Escherichia coli.

The gene encoding human alpha-1-antitrypsin (A1AT), when cloned and expressed as a full-length, non-fusion gene product in Escherichia coli, accumulates to levels up to 0.1% of total cellular protein. Truncation of the gene at its 5' end or synthesis as a fusion protein increases expression up to 200-fold. Extensive mutagenesis in vitro within this same 5'-terminal region aimed at improving codon usage and disrupting potential secondary structure increased expression only 10 to 20-fold. We have developed a translational fusion system for selecting mutations and applied it to the study of A1AT expression in E. coli. With this methodology, we have obtained single base-pair mutations having up to a 20-fold effect on A1AT expression. When we combined these multiple single base-pair mutations, we achieve up to a 200-fold increase in A1AT expression. The resulting gene product is of authentic size (394 amino acid residues) and contains two amino acid substitutions (Asn in place of Asp) in codons 2 and 6. This protein is primarily in the soluble fraction of the E. coli lysate and has identical activity to A1AT purified from human sera. The methodology used to generate these mutations may be generally applicable to the study of genes that do not express well in E. coli initially, and provides an alternative to secondary structure analysis in the redesign of such genes.

Amino Acid Sequence↗

PubMed1986

Quantitative Western blot assay for measurement of the murine acute phase reactant, serum amyloid P component.

Quantitation of the murine acute-phase reactant, serum amyloid P component (SAP), by Western blot is described. The assay is sensitive, reliable and inexpensive. Electrophoresis in standard SDS-polyacrylamide gels (SDS-PAGE) effectively separates SAP from other serum proteins. Electrophoretic blotting of SAP from the SDS-PAGE onto nitrocellulose (NC) paper is followed by a bovine serum albumin 'blocking' wash and exposure to anti-SAP antibody. Subsequent incubation with radioiodinated protein A was followed by autoradiography, and SAP bands were cut from the NC paper and counted in a gamma counter. The utility of this method for quantitation of SAP in biological fluids was verified using sera from normal mice and mice undergoing an acute inflammatory response. The results confirm the elevation of SAP associated with acute inflammation. The sensitivity of this technique coupled with the minute volumes of biological sample required renders it of potential utility for SAP quantitation in a variety of inflammatory disease states.

Amyloid↗

PubMed1985

Plasmid pFCE4: a new system of Escherichia coli expression-modification vectors.

Two versatile expression-modification vectors were obtained by inserting the origin of replication (ori) of phage f1 into the expression vector pOTS. The resulting plasmids produce large amounts of coding or noncoding ssDNA (depending on ori orientation in pFCE4+ and pFCE4-) and excrete it into the medium as virus-like particles following infection with phage f1. These features make them suitable for dideoxy chain termination sequencing, oligonucleotide directed mutagenesis and gene expression without further manipulations. The human IFN alpha-2 gene, lacking the codon for the first amino acid, cysteine, was efficiently expressed by these vectors.

Base Sequence↗