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PubMed1987

Construction of a cDNA clone containing the entire coding region of the human liver-type phosphofructokinase.

Genomic fragments containing coding sequences of the human liver phosphofructokinase (PFKL) were used to screen cDNA libraries and several clones corresponding to PFKL were isolated. Three overlapping cDNA clones spanning the entire coding region were stitched together yielding the plasmid pG-cPFKL3.0 containing a 3Kb PFKL cDNA down stream of the T7 promoter. To assess its coding capacity pG-cPFKL3.0 was transcribed by T7 RNA polymerase and the RNA product was used to program an in vitro translation system. An 80KDa polypeptide which co-migrated with an in vivo labeled PFKL and was recognized by anti PFKL antibodies was synthesized. These results show that the cDNA clone pG-cPFKL3.0 contains the entire coding region of the human PFKL.

Cloning, Molecular↗
PubMed1986

Advantages to mutagenesis techniques generating populations containing the complete spectrum of single codon changes.

The limitations of current mutagenesis techniques are analyzed in terms of the number and kinds of codon changes they make and in terms of the population size needed to produce all single or multiple amino acid variants. It is shown how a technique that can alter a single codon of a gene, producing all possible variant codons without affecting the rest of the gene, has certain advantages, if it can be used at each place in the gene in one experiment. Such a technique has advantages when the goals are to understand: (1) how specific structural alterations in a mutant protein cause it to function in a different but specific way, (2) how to predict which amino acids in a protein contact or interact with each other, and (3) why a protein is more or less sensitive to mutational disruption, depending upon the specific mutation. This is because it would generate the maximum number of (1) mutant proteins with different functions, (2) intracistronic suppressor for any starting mutation, and (3) random amino acid substitutions at random places. Furthermore, such a technique could produce useful variants more quickly and on a smaller scale than either evolution or current methods.

Amino Acids↗
PubMed2002

Enhanced DNA repair of cyclobutane pyrimidine dimers changes the biological response to UV-B radiation.

The goal of DNA repair enzyme therapy is the same as that for gene therapy: to rescue a defective proteome/genome by introducing a substitute protein/DNA. The danger of inadequate DNA repair is highlighted in the genetic disease xeroderma pigmentosum. These patients are hypersensitive to sunlight and develop multiple cutaneous neoplasms very early in life. The bacterial DNA repair enzyme T4 endonuclease V was shown over 25 years ago to be capable of reversing the defective repair in xeroderma pigmentosum cells. This enzyme, packaged in an engineered delivery vehicle, has been shown to traverse the stratum corneum, reach the nuclei of living cells of the skin, and enhance the repair of UV-induced cyclobutane pyrimidine dimers (CPD). In such a system, changes in DNA repair, mutagenesis, and cell signaling can be studied without manipulation of the genome.

Animals↗
PubMed1997

Plants as bioreactors for biopharmaceuticals: regulatory considerations.

Plants are one of several novel hosts that can be used for the production of recombinant biopharmaceuticals such as cytokines, hormones, monoclonal antibodies, enzymes and vaccines. The novelty of this technology and its wide range of potential applications require an assessment of possible regulatory concerns in the clinical development of plant-derived biopharmaceuticals. General principles extrapolated from experience gained with biotechnology products from other sources can serve as a foundation to develop scientifically sound strategies for the large-scale production and clinical development of safe and effective biopharmaceuticals in plant hosts.

Animals↗
PubMed2000

Reinventing the wheel.

Explore the source record for details and available documents.

Directed Molecular Evolution↗
PubMed1998

Pharmaceutical and therapeutic applications of artificial cells including microencapsulation.

Artificial cells for pharmaceutical and therapeutic applications started as microencapsulation on the micron scale. This has now expanded up to the higher range of macrocapsules and down to the nanometer range of nanocapsules and even to the macromolecular range of cross-linked hemoglobin as blood substitutes. This author first reported microencapsulation of biologically active material in 1957 (T.M.S. Chang, Hemoglobin corpuscles. Research Report for Honours Physiology, Medical Library, McGill University, 1957. (Also reprinted as part of 30th anniversary in Artificial Red Blood Cells Research, J. Biomater. Artif. Cells Artif. Organs 16 (1988) 1-9.) and 1964 (T.M.S. Chang, Semipermeable microcapsules, Science 146 (1964) 524-525). While pharmaceutical research has made use of these approaches for drug delivery, this author has been concentrating on the encapsulation of biotechnological products for therapeutic applications. Therefore, there was little interaction between the two approaches. In the last 10 years, pharmaceutical research, as in other areas of research, has become increasingly interested in biotechnology. Because of this interest, this article is a brief overview of developments of artificial cells for biotechnological products with emphasis on hemoglobin, enzymes, cells and genetically engineered microorganisms.

Animals↗
PubMed2002

Enzyme technology: an overview.

Enzymes are being used in numerous new applications in the food, feed, agriculture, paper, leather, and textiles industries, resulting in significant cost reductions. At the same time, rapid technological developments are now stimulating the chemistry and pharma industries to embrace enzyme technology, a trend strengthened by concerns regarding health, energy, raw materials, and the environment.

Biotechnology↗
PubMed2001

Exploring the impact of different thioesterase domains for the design of hybrid peptide synthetases.

BACKGROUND: A large number of pharmacologically important peptides are synthesized by multifunctional enzymes, the nonribosomal peptide synthetases (NRPSs). The thioesterase (Te) domain at the C-terminus of the last NRPS catalyzes product cleavage by hydrolysis or complex macrocyclization. Recent studies with excised Te domains and peptidyl-S-N-acetyl cysteamine substrate substitutes led to substantial insights in terms of cyclization activity and substrate tolerance of these enzymes. Their use in engineered hybrid NRPSs is an interesting but yet only little explored target for approaches to achieve new structural diversity and designed products. RESULTS: To study the capability of various Te domains to function in hybrid NRPSs, six different Te domains that catalyze different modes of termination in their natural systems were fused to a bimodular model NRPS system, consisting of the first two modules of tyrocidine NRPS, TycA and ProCAT. All Te domains were active in hydrolyzing the enzymatically generated dipeptide substrate D-Phe-Abu from the NRPS template with, however, greatly varying turnover rates. Two Te domains were also capable of hydrolyzing the substrate D-Phe-Pro and partially cyclized the D-Phe-Abu dipeptide, indicating that in an artificial context Te domains may display hydrolytic and cyclization activities that are not easily predictable. CONCLUSIONS: Te domains from heterologous NRPSs can be utilized for the construction of hybrid NRPSs. This is the first comparative study to explore their influence on the product pattern. The inherent specificity and regioselectivity of Te domains should allow control of the desired product cleavage, but can also lead to other modes of termination potentially useful for generating structural diversity. Our results provide the first data for choosing the proper Te domain for a particular termination reaction.

Chromatography, High Pressure Liquid↗
PubMed2003

Mitochondrial form of a tRNA synthetase can be made bifunctional by manipulating its leader peptide.

Previous studies showed that yeast VAS1 encodes both the cytoplasmic and mitochondrial forms of valyl-tRNA synthetase (ValRS), using alternative transcription and translation. The ValRS isoforms have identical polypeptide sequences, except for a 46-amino acid leader peptide that functions as a mitochondrial targeting signal. Although the two forms of the enzyme exhibit indistinguishable tRNA specificities in vitro, they cannot substitute for each other in vivo because of their different localizations. Here we show that the 46-residue leader sequence can be divided into two nonoverlapping peptides, each of which retains the ability to target the enzyme into mitochondria. The engineered proteins (with truncated leader sequences) are dual-targeted, rescuing both the cytoplasmic and mitochondrial defects of a vas1 knockout strain. Thus, in addition to alternative splicing and alternative translation initiation as mechanisms by which a single gene can encode cytoplasmic and mitochondrial activities, the inherent characteristics of a single polypeptide may enable it to be distributed simultaneously between two cellular compartments. This mechanism may explain how certain other single genes in Saccharomyces cerevisiae provide dual functions.

Cloning, Molecular↗
PubMed1999

Interchange of catalytic activity within the 2-enoyl-coenzyme A hydratase/isomerase superfamily based on a common active site template.

The structures and chemical pathways associated with the members of the 2-enoyl-CoA hydratase/isomerase enzyme superfamily are compared to show that a common active site design provides the members of this family with a CoA binding site, an expandable acyl binding pocket, an oxyanion hole for binding/polarizing the thioester C=O, and multiple active site stations for the positioning of acidic and basic amino acid side chains for use in proton shuttling. It is hypothesized that this active site template can be tailored to catalyze a wide range of chemical transformations through strategic positioning of acid/base residues among the active site stations. To test this hypothesis, the active site of one member of the 2-enoyl-CoA hydratase/isomerase family, 4-chlorobenzoyl-CoA dehalogenase, was altered by site-directed mutagenesis to include the two glutamate residues functioning in acid/base catalysis in a second family member, crotonase. Catalysis of the syn hydration of crotonyl-CoA, absent in the wild-type 4-chlorobenzoyl-CoA dehalogenase, was shown to occur with the structurally modified 4-chlorobenzoyl-CoA dehalogenase at kcat = 0.06 s-1 and Km = 50 microM.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMedDate not supplied

Conversion of fumaric acid to L-malic by sol-gel immobilized Saccharomyces cerevisiae in a supported liquid membrane bioreactor.

Conversion of fumaric acid (FA) to L-malic acid (LMA) was carried out in a bioreactor divided by two supported liquid membranes (SLMs) into three compartments: Feed, Reaction, and Product. The Feed/Reaction SLM, made of tri-n-octylphosphine oxide (vol 10%) in ethyl acetate, was selective toward the substrate, fumaric acid (S(FA/LMA) = 10). The Reaction/Product SLM, made of di(2-ethylhexyl) phosphate (vol 10%) in dichloromethane, was selective toward the product, L-malic acid (S(LMA/FA) = 680). Immobilized yeast engineered to overproduce the enzyme fumarase [E.C. 4.2.1.2] was placed in the Reaction compartment and served as the catalyst. The yeast was immobilized in small glasslike beads of alginate-silicate sol-gel matrix. The construction of the bioreactor ensured unidirectional flow of the substrate from the Feed to the Reaction and of the product from the Reaction to the Product compartments, with the inorganic counterion traveling in the opposite direction. The conversion of almost 100%, above the equilibrium value of ca. 84% and higher than that for the industrial process, 70%, was achieved. In contrast to the existing industrial biocatalytic process resulting in L-malic acid salts, direct production of the free acid is described.

Bioreactors↗