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R A Driedonks

Publications and source records attributed to R A Driedonks.

9 recordsLinked to original sources
PubMed1995

Expression and secretion of antifreeze peptides in the yeast Saccharomyces cerevisiae.

The antifreeze peptide AFP6 from the polar fish Pseudopleuronectus americanus has been expressed in and secreted by the yeast Saccharomyces cerevisiae as a biologically active molecule. The gene for the 37 amino acid long peptide has been chemically synthesized using yeast preferred codons. Subsequently, the gene has been cloned into an episomal expression vector as well as in a multicopy integration vector, which is mitotically more stable. The expression is under the control of the inducible GAL7 promoter. The enzyme alpha-galactosidase has been investigated as a carrier protein to facilitate expression and secretion of AFP. In order to reach increased expression levels, tandem repeats of the AFP gene (up to eight copies) have been cloned. In most cases the genes are efficiently expressed and the products secreted. The expression level amounts to approximately 100 mg/l in the culture medium. In a number of genetic constructs the genes are directly linked and expressed as AFP multimers. In other constructs linker regions have been inserted between the AFP gene copies, that allow the peptide to be processed by specific proteinases, either from the endogenous yeast proteolytic system or from a non-yeast source. The latter requires a separate processing step after yeast cultivation to obtain mature AFP. In all these cases proteolytic processing is incomplete, generating a heterogeneous mixture of mature AFP, carrier and chimeric protein, and/or a mixture of AFP-oligomers. The antifreeze activity has been demonstrated for such mixtures as well as for AFP multimers.

Amino Acid Sequence↗
PubMed1983

Gene 20 product of bacteriophage T4. II. Its structural organization in prehead and bacteriophage.

The location of gene 20 product of bacteriophage T4 in phage and phage percursors has been determined by immunochemical analysis of polyacrylamide gels, immunoturbidimetry and immunoelectron microscopy. The protein is present at the membrane attachment site of the prehead, a head precursor, and is accessible to the antibodies in the solution. It is present at the tail attachment site of the capsid, partially buried in the structure. In complete phage particles it is totally buried in the structure. It is in contact with the major shell proteins, gp23 and gp23*, respectively, in preheads and capsids, as revealed by partial crosslinking experiments. It forms the upper collar of the neck in necked tails. The lower collar is constructed from other gene products. On the basis of these data a structural model of the neck region of the phage has been derived. This model is consistent with a number of events in phage assembly, such as the role of gp20 in head assembly and DNA packaging, prehead detachment from the bacterial membrane and head-tail attachment. The symmetry mismatch known to occur between head and tail has been localized at the gp20-gp23* contact area.

Antibodies, Viral↗
PubMed1981

The quaternary structure of the T4 gene product 20 oligomer.

Gene product 20 of phage T4, which occurs in the phage particle near the head-tail interface, has been purified under moderate denaturing conditions. Upon renaturation the protein forms ring-like structures, sedimenting at 19 S. The rings measure 17 nm in diameter and 7 nm in height. They are characterized by a central hole of 3 nm and have 12-fold rotational symmetry. The data are consistent with a dodecameric structure with a molecular weight of 0.8 X 10(6) daltons. The oligomers are able to associate into double rings and occasionally into higher regular aggregates. It is thought that this structure initiates the assembly of the prehead core which serves as a scaffold for the assembly of the shell. This, as well as other implications of the gp20 structure are discussed.

Capsid↗
PubMed1978

Characterization of alfalfa-mosaic-virus protein polymerization in the presence of nucleic acid.

The polymerization of alfalfa mosaic virus (AMV) protein in the presence of homologous nucleic acids and a number of other natural and synthetic nucleic acids was studied. The conditions for optimal assembly were found to be pH 6.0 and low ionic strength (I = 0.1 M) at room temperature, irrespective of the type of nucleic acid. The resulting nucleoprotein particles exhibited the same structural characteristics as the virus. This information emerged from optical diffraction and computer filtering of electron micrographs from the reconstituted particles. Irrespective of the type of nucleic acid present the polymerization of the protein resulting in a nucleoprotein particle is a cooperative process. Evidence for this was obtained by nitrocellulose filter binding assay, sodium dodecylsulphate/polyacrylamide gel electrophoresis, sedimentation velocity and electron microscopy of the reaction mixtures. The rates and efficiencies of reconstitution were of the same order of magnitude for a number of ribonucleic acids. Sedimentation data derived from AMV protein and AMV RNA mixtures suggested the existence of a specific nucleation product in the first stage of assembly. The results are discussed in terms of a tentative model of the assembly, in which at least two different steps (nucleation and elongation) can be distinguished, each characterized by an association constant.

Chemical Phenomena↗
PubMed1976

A study of the states of aggregation of alfalfa mosaic virus protein.

The states of aggregation of alfalfa mosaic virus (AMV) protein have been characterized by sedimentation velocity experiments and electron microscopy. The main association product is a spherical particle with an s value of about 30S. It is highly likely that the assembly of this particle starts with dimers of the 25000 molecular mass unit resulting in an icosahedral particle made of 30 dimers. No intermediate aggregation products have been detected. The clustering pattern of the protein in the cylindrical part of the AMV capsid favours the concept of dimers as the active assembling units.

Capsid↗