Biomedical subjects

T Moks

Publications and source records attributed to T Moks.

25 records · Page 2Linked to original sources

PubMed1986

Secretion of heterologous gene products to the culture medium of Escherichia coli.

Different constructs containing fragments of the Staphylococcal protein A gene have been introduced in Escherichia coli and the effect on expression and translocation of the various heterologous gene products have been studied. By reversing the orientation of the different protein A gene constructions in the plasmid vector, a dramatic 20-fold difference in expression was obtained, accompanied with secretion of the gene product to the culture medium. Similar results were obtained by "heat-shock" treatment of the E.coli host cells. These results suggest the presence in the protein A gene of a stress induced promoter, functional in E.coli. The system was used to efficiently secrete a fusion protein consisting of a protein A fragment and human insulin-like growth factor I (IGF-I) to the culture medium of E.coli HB101. The fusion protein was purified from the culture medium by IgG affinity chromatography in a one-step procedure giving more than 95% yield.

Culture Media↗

PubMed1986

Staphylococcal protein A consists of five IgG-binding domains.

A genetic approach is described to clarify the IgG-binding properties of the N-terminal portion of staphylococcal protein A (region E). Several gene fragments, encoding region E or B or protein A, have been cloned and expressed in Escherichia coli. The gene products were purified by IgG-affinity chromatography and subjected to structural and functional analyses. Both fragments can be efficiently purified using this method, suggesting that region B as well as region E has Fc-binding activity. In addition, gene fusions were assembled giving fragments EB and EE, which both showed a divalent Fc-binding. These results demonstrate that protein A consists of five IgG-binding domains. The implications of these findings for the structure of protein-A--immunoglobulin-G complexes are discussed.

Amino Acids↗

PubMed1986

Production of specific antibodies against protein A fusion proteins.

The gene for Staphylococcal protein A was fused to the coding sequence of bacterial beta-galactosidase, alkaline phosphatase and human insulin-like growth factor I (IGF-I). The fusion proteins, expressed in bacteria, were purified by affinity chromatography on IgG-Sepharose and antibodies were raised in rabbits. All three fusion proteins elicited specific antibodies against both the inserted protein sequences and the protein A moiety. In the case of IGF-I, the protein A moiety in the fusion protein may act as an adjuvant since native IGF-I alone is a poor immunogen. The results suggest that the protein A fusion system can be used for efficient antibody production against peptides or proteins expressed from cloned or synthetic genes. To facilitate such gene fusions a set of optimized vectors have been constructed.

Alkaline Phosphatase↗

PubMed1985

Analysis of signals for secretion in the staphylococcal protein A gene.

Different constructs of the gene encoding staphylococcal protein A were introduced in Staphylococcus aureus and S. xylosus as well as Escherichia coli. The product of the gene without the cell wall anchoring domain was efficiently secreted in all three hosts. N-terminal sequencing of the affinity-purified mature protein revealed a common processing site after the alanine residue at position 36. In contrast, when an internal IgG-binding fragment of protein A (region B) was inserted after the protein A signal sequence, the product was poorly secreted and N-terminal sequencing revealed no processing at the normal site. This demonstrates that the structure of the polypeptide chain beyond the signal peptide cleavage site can affect cleavage. Another construct, containing the N-terminal IgG-binding part of the mature protein A (region E) followed by region B, gave correct processing and efficient secretion. Unexpectedly, the gene product, EB, was not only secreted and correctly processed, but was also excreted to the culture medium of E. coli. Secretion vectors containing the protein A signal sequence were constructed to facilitate secretion of foreign gene products. Insertion of the E. coli gene phoA, lacking its own promoter and signal sequence, led to efficient secretion of alkaline phosphatase both in E. coli and S. aureus.

Amino Acid Sequence↗

PubMed1984

Uniformly spaced banding pattern in DNA sequencing gels by use of field-strength gradient.

A method is described for casting and operating ultrathin wedge-shaped field-strength gradient gels for DNA sequencing. One of the gradients results in a uniformly-spaced oligonucleotide banding pattern from 60 to 300 nucleotides in a 53 cm long 4% polyacrylamide gel. From these field-strength gradient gels, more sequencing data can be obtained with greater confidence than from gels with uniform thickness. The gel-casting method is fast and simple. At 2.5 kV two gels can be run simultaneously in 2 h with the same power supply.

Bacillus↗

PubMedDate not supplied

Semi-automated solid-phase DNA sequencing.

Increasing the efficiency of DNA sequencing necessitates the development of systems which reduce the need for manual operations by integrating template preparation, sequencing reactions, product separation and detection. A semi-automated system, whereby PCR-amplified biotinylated genomic or plasmid DNA is immobilized on streptavidin-coated magnetic beads, has been developed.

Automation↗

PubMedDate not supplied

A synthetic IgG-binding domain based on staphylococcal protein A.

A synthetic IgG-binding domain based on staphylococcal protein A was designed with the aid of sequence comparisons and computer graphic analysis. A strategy, utilizing non-palindromic restriction sites, was used to overcome the difficulties of introducing site-specific changes into the repetitive gene. A single mutagenized gene fragment was polymerized to different multiplicities, and the different gene products were expressed in Escherichia coli. Using this scheme, protein A-like proteins composed of different numbers of IgG-binding domains were produced. These domains were changed to lack asparagine--glycine dipeptide sequences as well as methionine residues and are thus, in contrast to native protein A, resistant to treatment with hydroxylamine and cyanogen bromide.

Amino Acid Sequence↗