Versatile expression system for rapid and stable production of recombinant proteins.

Previously we reported the development of a novel expression system with Tat/TAR-oriP vectors and HKB11 cell line, which supports high level protein expression (Cho et al. Cytotechnology 2001, 37, 23-30). In the present study, we further demonstrated that HKB11 cells are suitable for high throughput expression (microgram scale) of genomic candidates in transient transfection system for in vitro evaluation of biological functions. HKB11 cells were also shown to support the production of milligram to gram quantities of protein drug candidates for in vivo evaluation of efficacy in various disease models. Stable HKB11 clones secreting high levels of a tissue factor (TF; 40-50 pg/c/d) and B-domain deleted recombinant factor VIII (BDDrFVIII; 5-10 microU/c/d) were derived under serum-free conditions. The specific productivity for these two proteins from the HKB11 cells was 10-fold greater than those from CHO cells derived under the similar conditions. In conclusion, we have demonstrated that the HKB11 cell line is well-suited for transient and long-term production of recombinant proteins.

Prions and nucleic acids: search for "residual" nucleic acids and screening for mutations in the PrP-gene.

Studies on prions involve nucleic acid chemistry under two headings: i) do infectious prion particles contain nucleic acids? ii) is it possible by a simple procedure to screen the prion protein (PrP)-gene for mutations? The return refocusing gel electrophoresis technique was developed to detect by sensitive silver staining homogeneous and heterogeneous nucleic acids extracted from highly purified scrapie prion preparations. With this method all kinds of nucleic acids from a length of 13 nucleotides up to several thousand could be recovered and detected with a yield over 90%. Despite extensive nuclease digestion some small polynucleotides remained. The results define clear restrictions for a hypothetical scrapie-specific nucleic acid. If homogeneous in size, such a molecule would be smaller than 80 nucleotides in length at a particle-to-infectivity ratio near unity; if heterogeneous, scrapie-specific nucleic acids would have to include molecules smaller than 240 nucleotides. To detect mutations in the PrP-gene, either known mutations from human prion diseases or artificial ones in transgenic animals, or to screen for not yet identified mutations in patients, a method is required which guarantees detection of mutations which might occur in every single position of the whole PrP-ORF. It will be shown that a combination of PCR and temperature-gradient gel electrophoresis fulfils these requirements. By thermodynamic calculations the shift in the gel electrophoresis due to a mutation can be calculated depending on the position of the mutation. The theoretical results were tested with the mutations known so far.

Analysis of nucleic acids in purified scrapie prion preparations.

Amount, type, and size of nucleic acid molecules associated with purified prion preparations were analyzed. Return refocusing gel electrophoresis (RRGE) was developed to detect homogeneous and heterogeneous nucleic acids extracted from highly purified scrapie prion preparations. With this method all types of nucleic acids in the size range from 13 to several thousand nucleotides could be analyzed. The recovery of all nucleic acids, after deproteinization and two-phase extraction was higher than 90%. Despite extensive nuclease digestions some small polynucleotides remained. Although a scrapie-specific nucleic acid cannot be excluded, the results further define the possible characteristics for such a hypothetical molecule. If it was homogeneous in size, then it would be < 80 nt in length at a particle-to-infectivity ratio (P/I) near unity; if the other extreme, i.e. totally heterogeneous scrapie-specific nucleic acids were assumed, then scrapie-specific nucleic acids would have to include molecules smaller than 240 nt. In order to exclude the possibility that unspecific background nucleic acid is entrapped in prion-rods, infectious material has to be prepared without a proteolysis and rod formation, and the analysis of nucleic acids performed with those preparations.

Further analysis of nucleic acids in purified scrapie prion preparations by improved return refocusing gel electrophoresis.

Although increasingly unlikely, the possibility of a scrapie-specific nucleic acid carried by infectious prion particles is still unresolved. Return refocusing gel electrophoresis was developed to detect homogeneous and heterogeneous nucleic acids extracted from highly purified scrapie prion preparations. This method was improved with respect to the size range from 13 to 1100 nucleotides (nt) over which analyses could be performed. The yield of nucleic acid, particularly of small DNA oligonucleotides and polyadenylated RNA, was determined after deproteinization and two-phase extraction. Despite extensive nuclease digestions some small polynucleotides remained. Although a scrapie-specific nucleic acid cannot be excluded, the results further define the possible characteristics of a hypothetical molecule. If homogeneous in size, such a molecule would be less than 80 nt in length at a particle-to-infectivity ratio near unity, if heterogeneous, scrapie-specific nucleic acids would have to include molecules smaller than 240 nt.

Paradoxical shortening of scrapie incubation times by expression of prion protein transgenes derived from long incubation period mice.

Prolonged incubation times for experimental scrapie in I/LnJ mice are dictated by a dominant gene linked to the prion protein gene (Prn-p). Transgenic mice were analyzed to discriminate between an effect of the I/LnJ Prn-pb allele and a distinct incubation time locus designated Prn-i. Paradoxically, 4 independent Prn-pb transgenic mouse lines had scrapie incubation times shorter than nontransgenic controls, instead of the anticipated prolonged incubation periods. Aberrant or overexpression of the Prn-pb transgenes may dictate abbreviated incubation times, masking genuine Prn-p/Prn-i congruence; alternatively, a discrete Prn-i gene lies adjacent to Prn-p.

Search for a putative scrapie genome in purified prion fractions reveals a paucity of nucleic acids.

Scrapie can be transmitted by novel infectious pathogens termed prions. No evidence for a scrapie-specific nucleic acid has been detected to date. To investigate amounts, types and sizes of nucleic acid molecules associated with prions in purified preparations, aliquots were deproteinized, and the nucleic acids analysed by PAGE and silver staining. Digestion with nucleases and exposure to Zn2+ prior to analysis substantially diminished the content of nucleic acids, but did not alter the prion titre indicating that those nucleic acids which were removed are not essential for infectivity. Since a single species of scrapie-specific nucleic acid could not be identified, we explored the unprecedented possibility of scrapie-specific nucleic acids of variable length which are biologically active. If such molecules of variable length exist then they might be hidden within the background smear on silver-stained gels after PAGE. A new procedure designated return refocusing gel electrophoresis (RRGE) was developed to identify heterogeneous nucleic acids in purified prion fractions. The content of variable length nucleic acids was reduced by a factor of 10 by exhaustive Bal 31 exonuclease digestion after dispersion of purified prions into detergent-lipid-protein complexes. For example, a typical sample after Bal 31 digestion contained approximately 4 ng of nucleic acid of variable length and 10(8.7) ID50 units of scrapie prion infectivity. Consideration of different models for a hypothetical scrapie-specific nucleic acid suggests that such a molecule would have to be: (i) quite small (less than 100 nucleotides), (ii) possess a particle-to-infectivity ratio near unity or (iii) heterogeneous in size. Although our results do not eliminate the possibility that prions possess a scrapie-specific nucleic acid of variable length, they narrow considerably the spectrum of features specifying such a candidate molecule.

Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication.

Transgenic (Tg) mice expressing both Syrian hamster (Ha) and mouse (Mo) prion protein (PrP) genes were used to probe the mechanism of scrapie prion replication. Four Tg lines expressing HaPrP exhibited distinct incubation times ranging from 48 to 277 days, which correlated inversely with HaPrP mRNA and HaPrPC. Bioassays of Tg brain extracts showed that the prion inoculum dictates which prions are synthesized de novo. Tg mice inoculated with Ha prions had approximately 10(9) ID50 units of Ha prions per gram of brain and less than 10 units of Mo prions. Conversely, Tg mice inoculated with Mo prions synthesized Mo prions but not Ha prions. Similarly, Tg mice inoculated with Ha prions exhibited neuropathologic changes characteristic of hamsters with scrapie, while Mo prions produced changes similar to those in non-Tg mice. Our results argue that species specificity of scrapie prions resides in the PrP sequence and prion synthesis is initiated by a species-specific interaction between PrPSc in the inoculum and homologous PrPC.

Transgenic mice expressing hamster prion protein produce species-specific scrapie infectivity and amyloid plaques.

Three transgenic mouse lines designated Tg 69, 71, and 81 were produced harboring a Syrian hamster (Ha) prion protein (PrP) gene; all expressed the cellular HaPrP isoform in their brains. Inoculation of Tg 81 mice or hamsters with Ha prions caused scrapie in integral of 75 days; nontransgenic control mice failed to develop scrapie after greater than 500 days. Tg 71 mice inoculated with Ha prions developed scrapie in integral of 170 days. Both Tg 71 and Tg 81 mice exhibited spongiform degeneration and reactive astrocytic gliosis, and they produced the scrapie HaPrP isoform in their brains. Tg 81 brains also showed HaPrP amyloid plaques characteristic of Ha scrapie and contained integral of 10(9) ID50 units of Ha prions based on Ha bioassays. Our findings argue that the PrP gene modulates scrapie susceptibility, incubation times, and neuropathology; furthermore, they demonstrate synthesis of infectious scrapie prions programmed by a recombinant DNA molecule.

Distinct prion proteins in short and long scrapie incubation period mice.

The Prn-i gene, controlling scrapie incubation period, is linked to or congruent with the murine prion protein (PrP) gene, Prn-p. In prototypic mouse strains with long (l/Ln) and short (NZW) incubation periods, Prn-p transcription is initiated at similar multiple sites. The predicted NZW and l/Ln PrP proteins differ at codons 108 and 189. Codon 189, highly conserved in mammals, lies within a polymorphic BstEll site that is retained in 17 mouse strains known to have short or intermediate incubation times, but is absent in l/Ln and two other inbred mice with long incubation times. Codon 108 in mice with short or intermediate incubation times encodes Leu; in mice with long incubation times it encodes Phe. The correlation of PrP sequence with length of scrapie incubation period suggests, but does not formally prove, congruency between Prn-p and Prn-i.

Molecular cloning of Friend mink cell focus-inducing virus: identification of mink cell focus-inducing virus-like messages in normal and transformed cells.

Friend mink cell focus-inducing virus (Fr-MCF) is a leukemogenic murine retrovirus that was isolated from a leukemic NIH Swiss mouse. We molecularly cloned the genome of this virus into pBR322. Restriction enzyme mapping of this cloned Fr-MCF DNA revealed a 1.0-kilobase-pair region in the envelope gene which differs from the restriction map of the Friend ecotropic virus (Friend murine leukemia virus). A 400-base-pair Fr-MCF envelope gene fragment was subcloned from this region and designated pLEK. Probes made with pLEK hybridize to Fr-MCF DNA but do not hybridize to either Friend or amphotropic murine leukemia virus DNA. Polyadenylic acid-selected RNA was prepared from the hematopoietic tissues of normal NIH Swiss mice, Fr-MCF-infected erythroleukemia cells (TP-1), and uninfected chemically transformed T lymphocytes (RB-1). The pLEK probe identified 34S and 22S messages in the TP-1 cells and in the normal hematopoietic tissues. RB-1 cells contain 32S and 26S messages that hybridize to pLEK. However, the pLEK-like RNAs found in the normal hematopoietic tissues and in the RB-1 cells were 400-fold less abundant than the RNAs found in the TP-1 cells.