Georg Sczakiel

EXPRESSION OF P21 PROTEINS IN ESCHERICHIA-COLI AND STEREOCHEMISTRY OF THE NUCLEOTIDE-BINDING SITE

v‐Ha‐ras encoded p21 protein (p21V), the cellular c‐Ha‐ras encoded protein (p21C) and its T24 mutant form p21T were produced in Escherichia coli under the control of the tac promoter. Large amounts of the authentic proteins in a soluble form can be extracted and purified without the use of denaturants or detergents. All three proteins are highly active in GDP binding, GTPase and, for p21V, autokinase activity. Inhibition of [3H]GDP binding to p21C by regio‐ and stereospecific phosphorothioate analogs of GDP and GTP was investigated to obtain a measure of the relative affinities of the three diphosphate and five triphosphate analogs of guanosine. p21 has a preference for the Sp isomers of GDP alpha S and GTP alpha S. It has low specificity for the Sp isomer of GTP beta S. Together with the data for GDP beta S and GTP gamma S these results are compared with those obtained for elongation factor (EF)Tu and transducin. This has enabled us to probe the structural relatedness of these proteins. We conclude that p21 seems to be more closely related to EF‐Tu than to transducin.

Spontaneous uptake of biologically active recombinant DNA by mammalian cells via a selected DNA segment

DNA can be internalized by mammalian cells without taking advantage of helper reagents. Here, we ask whether the spontaneous cellular uptake of double-stranded DNA (dsDNA) occurs in a biologically significant and sequence-dependent way. We describe a combinatorial approach to search for dsDNA sequence segments that are preferentially internalized. A selected dsDNA species was identified and covalently linked to a luciferase expression cassette. The increased apparent cellular uptake of long-chain recombinant DNA accompanied by an increased apparent expression of luciferase provides strong evidence for the view that (i) naked long-chain dsDNA can be taken up spontaneously by mammalian cells, (ii) specific sequences substantially increase this process, and (iii) dsDNA is transported into the nucleus of cells in a bioactive form. Experimental evidence indicates a tissue- or cell-type specificity for this process. This work indicates that, in principle, specific nucleotide sequences can facilitate the introduction of naked dsDNA into target cells of interest, thereby improving existing vector systems and providing a new methodology to study DNA uptake by mammalian cells. The cellular uptake of biologically active genetic material in vivo occurs to be conceivable.

EXPRESSION OF NATIVE RABBIT LIGHT-MEROMYOSIN IN ESCHERICHIA-COLI - OBSERVATION OF A POWERFUL INTERNAL TRANSLATION INITIATION SITE

The cDNA-sequence coding for rabbit skeletal muscle light meromyosin (LMM) was placed under the control of the lambda promoter (PL) of an Escherichia coli expression vector. The resulting plasmid pEXLMM74 expressed non-fused rabbit skeletal muscle LMM with yields ranging from 1 to 5% of the total proteins of E. coli. This LMM was specifically recognized by polyclonal antibodies raised against chicken pectoralis muscle myosin. It could be highly enriched from E. coli extracts by using two cycles of high and low ionic strength buffer. The partially purified protein contained a major side-product, with a calculated molecular mass of 59 kilodaltons, that is produced by translation initiation from a site in the coding region of LMM. After deletion of the translation initiation site derived from the expression plasmid, only the 59 kilodalton protein is expressed in E. coli from the resulting plasmid pEXLMM59. Both the 74 and 59 kilodalton proteins were shown to form paracrystals. They were studied by electron microscopy using negative staining and were found to show characteristic striations with an axial periodicity of about 43 nm. By circular dichroism measurement we showed that the purified 59 kilodalton protein is folded mostly as an alpha-helix.

Selected Strategies for the Delivery of siRNA In Vitro and In Vivo

RNA-based therapeutic strategies are considered as a highly promising alternative to conventional drug development. Among the different classes of oligonucleotide-derived prospective drugs, small interfering RNAs (siRNAs) are of particular interest. However, cellular uptake and subsequent intracellular trafficking to the effector complex (RNA-induced silencing complex; RISC) represent major technical hurdles for the efficacy of these macromolecular drugs. Thus, the development of appropriate delivery systems is an essential requirement to turn these molecules into medicine. In this review, we will focus on two particular auspicious aspects in this context, the phosphorothioate-stimulated uptake of naked siRNA and the use of cell-penetrating peptides as shuttles for a controlled cellular uptake. Moreover, we will present some of the most promising recent approaches for siRNA delivery in vivo, which may help to pave the road to drugs of the future.

31P-NMR spectra of the Ha-ras p21. Nucleotide complexes

Phosphorus nuclear magnetic resonance spectra of the Ha-ras oncogene product p21 and its nucleotide complexes have been obtained. It is shown that the 31P nuclear magnetic resonance spectra of a number of nucleotide-enzyme complexes show some common features. In particular, the chemical shift values of the beta-phosphorus resonance of enzyme-bound NTP and NDP (N = A, G) of hydrolases exhibit a downfield shift virtually identical for myosin, elongation factor Tu, and the Ha-ras oncogene product p21. This suggests that the stereochemistry around the beta-phosphorus might be similar in these compounds.

Vector pPLEX for expression of nonfusion polypeptides in Escherichia coli

Publisher Summary Escherichia coli ( E. coli ) bacteria are a powerful tool for the production of heterologous proteins in large quantities, which is of general experimental importance in many fields of natural sciences. E. coli are one of the best-studied organisms and many well-established methodologies used in molecular biology can be applied to modify and handle vectors and coding sequences. Critical parameters for successful production of pPLEX-encoded proteins include the conditions of induction, that is, the time period and temperature of heat shock. As the induction of the αp L promoter usually is mediated by a temperature shift to 42 °, the heat-shock response of E. coli cells, which is accompanied by induced expression of E. coli proteases, can affect the stability of expressed proteins. In addition, the time period of induction determines the accumulation of expression products, which has a crucial effect on yields and the physical form of the expression products. One of the more recent improvements of pPLEX was the insertion of additional restriction sites between the SalI and BclI sites, creating the modified vector pPLEX.

[17] – Vector pPLEX for Expression of Nonfusion Polypeptides in Escherichia coli

Publisher Summary nEscherichia coli bacteria is a powerful tool for the production of heterologous proteins in large quantities, which is of general experimental importance in many fields of natural sciences such as biochemical and biophysical studies. The functional genes coding for polypeptides are introduced stably into E. coli bacteria by E. coli vectors. Fusion proteins can be used for immunological studies, such as the production of antisera, or as antigens in enzyme-linked immunosorbent assay or western analysis. However, their use in other studies which concerns enzymatic activities and three-dimensional structures, is limited to where the expression of nonfusion proteins is desirable. There are a number of other parameters for successful expression of heterologous eukaryotic sequences in E. coli: (1) DNA sequence and primary and secondary structure of the transcript in the vicinity of the start codon, (2) codon usage, (3) possible toxicity of expression products for E. coli, (4) posttranslational modifications, (5) RNA editing of eukaryotic sequences in the homologous system, which does not occur in E. coli, and (6) evaluation of the ability of expressed portions of proteins to form defined structures.

Applied Tumor Virology

The Research Program Applied Tumor Virology investigates the role of various viruses in carcinogenesis.