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HUMAN GLYCOPROTEINS AND DERIVED VARIANTS FROM RECOMBINANT MAMMALIAN CELLLINES
No full textThe expression of foreign genes using recombinant DNA technology in various host systems has
permitted the production of human proteins of therapeutic interest in high amounts. Manyclinically
important human proteins are posttranslationally modified. However, the inability of microbes
to perform mammalian-type of posttranslational modifications of proteins is a major
shortcoming. Alternative expression systems are insect and mammaliancells. Principle mammalian
types of protein modifications are N- and O-glycosylation. Insect cells, fungi and yeasts
are unable to perform the same terminal glycosylation reactions on glycoproteins as mammalian
cells. Recombinant DNA technology used for the production of pharmaceutically useful polypeptides
has mainly been focused on microbial expression systems (bacteria like E. coli, yeast and
fungi). The advantage of microbial expression systems is the high amount of expressed protein
that can be obtained.
The present communication considers aspects of glycoprotein research relevant to the field of
biotechnology and protein design. Results are presented that have been obtained by our group
during the last four years concerning the expression of the glycoproteins human Interleukin 2
(Il-2) and Interferon-8 (IFN-8) in different mammalian cell lines, the determination of their
carbohydrate residues, the effect of site-directed mutagenesis on their carbohydrate attachment
sites and the insertion of peptide domains which function as acceptors for carbohydrates
THE CROSSOVER LINKER. MECHANISMS AND APPLICATIONS IN GENE MODIFICATION
No full textWe have developed a novel method for mutating DNA sequences, based on site-specific, in
vivo, recombination, known as the crossover linker method. A typical crossover linker
contains; (i) a single-stranded overhang for an initial cohesive-end ligation with one terminus
of a linearized plasmid, (ii) a mid-section carrying modified sequence information, and (iii) a
"homology-searching" sequence at the other end, that is similar to a specific region in the
opposite terminus of the plasmid. Following transformation of an E. coli host with a
plasmid/linker complex, intramolecular recombination between the homologous regions of
the resultant intermediate completes the circularization of the plasmid, with concomitant
integration of the linker. Crossover linking is performed on double-stranded DNA and can be
used to create deletions andinsertions, as well as to perform site-specific mutagenesis. Both
single- and double-stranded linkers with "homology searching" region as short as 5 nucleotides
can be used for gene modification. Deletions of over 1000 bp have been achieved using
"homology searching" regions of approx. 20 nucleotides in length. In this article, the
effectiveness, limitations and mechanism of this process are discussed with emphasis on the
application of the crossoverlinker to the manipulation of protein-encoding sequences
NEUE MÖGLICHKEITEN ZUR ENZYMATISCHEN HERSTELLUNG VON L-PHENYLALANIN
No full textThree enzyme-catalyzed processes for the production of L-phenylalanine
are presented, starting with phenylpyruvate, D,L-phenyllactate, or
acetamidocinnamate, respectively. In each case, the amino acid is
formed by reductive amination catalyzed by the L-phenylalanine
dehydrogenase. Space-time-yields up to 456 g/l-d can be reached using
continuously operating processes
TOWARDS THE CONSTRUCTION OF NEW PROTEINS
No full textThe construction of new proteins is a challenging goal in present peptide and
protein chemistry (2). Success in the chemical synthesis of de novo designed
small proteins has been limited until now because of our still limited knowledge
of the factors that determine the folding of a polypeptide chain. We have
proposed a new strategy which aims at avoiding this problem by the use of the
specific possibilities of peptide chemistry, for the synthesis of template—
assembled synthetic proteins (TASPs)
CHARACTERISATION OF ENGINEERED PROTEINS: SOME CRITICAL REFLECTIONS
No full textThis essay is an attempt to point up the gap between, on the one hand, the
methods currently available to the biologist in the laboratory and, on the other,
the kind of data that he or she would need in order to characterise genetically
engineered proteins of topical biological interest in such a way as to make use
of the techniques of protein engineering.
Sgren Kirkegaard was Denmark's greatest philosopher, and he was well aware of the
fact. One day he reflected: “To be Denmark's greatest philosopher, ah, that is indeed a
fine satire.” By this he presumably meant that he was the only one. These words have
encouraged us to philosophize a little about the protein engineering cycle, of which our
version is shownin Figure 1.
We have dissected the cycle according to two principles, information-theoretical
(vertical axis) and epistemological (horizontal axis). The cycle starts from a gene and
proceeds via expression to the corresponding protein, which we associate with a set of
properties by testing or suitable characterisation. The understanding of these leads by
way of theory, experience or intuition to a new gene, and thereafter the cycle continues, a
process of which we have seen many impressive examples
DEVELOPMENT OF HIGH SENSITIVE AMPEROMETRIC ENZYME ELECTRODES
No full textHigh sensitive amperometric enzyme electrodes based on chemical
emplification in biocatalytic membranes or biocatalytic accumulation
of oxidation-reduction equivalents on chemically modified electrodes
have been designed.
Chemical amplification via cyclic substrate conversion in biocatalytic
membranes proceeds merely at high diffusion moduli values.
The amplification degree depends on the catalytic activity of membranes
and their thickness as well. The response of the electrodes
with cyclic substrate conversion is slow.
High sensitive determination of aromatic amines was carried out
with the use of enzyme electrodes in the biocatalytic layer of which
the substrate-substrate activation of peroxidase catalysis occurs.
The activation in the bienzyme glucose oxidase/peroxidase system is
observed at high glucose concentrations.
Biocatalytic product accumulation, accomplished through the apoenzyme
and cofactor interaction, enables to determine a substance in
concentration relative to the dissociation constant of choloenzyme.
The accumulation of oxidation-reduction equivalents on chemically modified
electrodes with their subsequent electrochemical conversion
makes the basis for stripping analysis, ascertaining the determination
of low metabolite concentrations
NEW TRENDS ON MODIFIED ELECTRODES USED IN THE BIOMEDICAL AND PHARMACEUTICAL FIELD
No full textThe purpose of this work is to illustrate the increasing interest of biosensors and
modified electrodes in the field of pharmaceutical potentiometric and voltammetric
analysis. Two amino-acids have been determined using enzyme or bacterial membranes
immobilized on gas sensing electrodes. The analytical parameters involved in the
optimization of the electrode response, but also microbiological factors are discussed
for each electrode. Performances of the electrodes and interferences which may occur
are presented. Opportunities of using bacteria to produce specific unstable enzymes
are pointed out. Neuroleptics have been potentiometrically determined using polymeric
modified electrodes specific to drugs containing a piperazine, piperidine or pyrrolidine
group.
Some new trends in electrode modification have been developed with special emphasis
on voltammetric applications. Physical surface modification of carbon paste electrodes
by incorporation of an appropriate catalyst (metalloporphyrine derivative) into the
electrode substrate has permitted to analyse several pharmaceutical interesting
compounds which exhibit poor voltammetric curves at unmodified electrodes. Electrochemical
activation of carbon paste electrodes by electrodeposition of platinum microparticles
has extended the performances of this type of sensor, with regard to
sensitivity, for the investigation of platinum (II) based antitumor complexes. These
modification techniques offer benefits for the quantitative analysis of organic
compounds but permit also to investigate more deeply the mechanistic side of the
interaction electrode - molecule
SYNTHESIS AND CHARACTERIZATION OF A SET OF FOUR DODECADEOXYRIBONUCLEOSIDE UNDECAPHOSPHATES CONTAINING 0°-METHYLGUANINE OPPOSITE ADENINE, CYTOSINE, GUANINE, AND THYMINE
No full textA set of four self-complementary dodecanucleoside undecaphosphates,
d[CGNGAATTC(O6Me)GCG] (1), where N = A, C, G, or T, has been synthesized by a phosphoramidite
procedure. Each sequence forms a stable duplex, with a Im between 19
and 26°C lower than the Tm Of the "parent" molecule d(CGCGAATTCGCG). The lowest
melting sequence is the N=T molecule; the overall order is N = C>A>G> T. Enus
0°-met hylation of guanine creates a region of localized instability in DNA regardless
of the base opposite the lesion
APPLICATIONS OF SYNTHETIC PEPTIDES
No full textThe revolutionary development of molecular biology during the past
years has led to a strong interest in synthetic peptides. In this
talk several important applications of synthetic peptides are discus
sed.
Peptides can be synthesized chemically in solution (1) or on solid
supports (2,3). Up to a length of approximately 50 residues synthetic
peptides have been obtained in (nearly) homogeneous form (4-6)
PROTEIN ENGINEERING BY SITE DIRECTED MUTAGENESIS
No full textThe construction of mutations in the active site of the tyrosyl tRNA
synthetase from Bacillus stearothermophilus has allowed us to deduce the
relative impörtance of the substrate contacts to transition state binding.
The feature dominating the energetics is the exchange reaction with water
molecules: thus by deleting a poor H-bonding contact to the substrate we
could increase the affinity of the enzyme for substrate. Furthermore by
straining the polypeptide backbone by introducing a proline residue, we
could improve the interaction of a histidine residue with the substrate.
Thus enzymes affinities can be bettered by protein engineering in vitro