Protein secretion biotechnology using Streptomyces lividans: large-scale production of functional trimeric tumor necrosis factor alpha.

We evaluated the feasibility of large-scale production of biopharmaceuticals expressed as heterologous polypeptides from the Gram-positive bacterium Streptomyces lividans. As a model protein we used murine tumor necrosis factor alpha (mTNFalpha). mTNFalpha fused C-terminally to the secretory signal peptide of the subtilisin-inhibitor protein from Streptomyces venezuelae. Under appropriate fermentation conditions, significant amounts of mature mTNFalpha (80-120 mg/L) can be recovered from spent growth media. Efficient downstream processing allowing rapid purification of mTNFalpha from culture supernatants was developed. Importantly, the protein is recovered from the spent growth medium in its native trimeric state as judged by biophysical analysis. Further, mTNFalpha secreted by S. lividans is significantly more active in an in vitro apoptosis tissue culture assay than a corresponding polypeptide produced in Escherichia coli. This pilot study provides the first validation of S. lividans protein secretion as an alternative bioprocess for large-scale production of oligomeric proteins of potential therapeutic value.

Cross-talk between catalytic and regulatory elements in a DEAD motor domain is essential for SecA function.

SecA, the motor subunit of bacterial polypeptide translocase, is an RNA helicase. SecA comprises a dimerization C-terminal domain fused to an ATPase N-terminal domain containing conserved DEAD helicase motifs. We show that the N-terminal domain is organized like the motor core of DEAD proteins, encompassing two subdomains, NBD1 and IRA2. NBD1, a rigid nucleotide-binding domain, contains the minimal ATPase catalytic machinery. IRA2 binds to NBD1 and acts as an intramolecular regulator of ATP hydrolysis by controlling ADP release and optimal ATP catalysis at NBD1. IRA2 is flexible and can undergo changes in its alpha-helical content. The C-terminal domain associates with NBD1 and IRA2 and restricts IRA2 activator function. Thus, cytoplasmic SecA is maintained in the thermally stabilized ADP-bound state and unnecessary ATP hydrolysis cycles are prevented. Two DEAD family motifs in IRA2 are essential for IRA2-NBD1 binding, optimal nucleotide turnover and polypeptide translocation. We propose that translocation ligands alleviate C-terminal domain suppression, allowing IRA2 to stimulate nucleotide turnover at NBD1. DEAD motors may employ similar mechanisms to translocate different enzymes along chemically unrelated biopolymers.

A molecular switch in SecA protein couples ATP hydrolysis to protein translocation.

SecA, the dimeric ATPase subunit of bacterial protein translocase, catalyses translocation during ATP-driven membrane cycling at SecYEG. We now show that the SecA protomer comprises two structural modules: the ATPase N-domain, containing the nucleotide binding sites NBD1 and NBD2, and the regulatory C-domain. The C-domain binds to the N-domain in each protomer and to the C-domain of another protomer to form SecA dimers. NBD1 is sufficient for single rounds of SecA ATP hydrolysis. Multiple ATP turnovers at NBD1 require both the NBD2 site acting in cis and a conserved C-domain sequence operating in trans. This intramolecular regulator of ATP hydrolysis (IRA) mediates N-/C-domain binding and acts as a molecular switch: it suppresses ATP hydrolysis in cytoplasmic SecA while it releases hydrolysis in SecY-bound SecA during translocation. We propose that the IRA switch couples ATP binding and hydrolysis to SecA membrane insertion/deinsertion and substrate translocation by controlling nucleotide-regulated relative motions between the N-domain and the C-domain. The IRA switch is a novel essential component of the protein translocation catalytic pathway.

The formation of new nucleoli during macronuclear development of the hypotrichous ciliate Stylonychia lemnae visualized by in situ hybridization.

After sexual reproduction in ciliates, the old macronucleus degenerates and a new macronucleus is formed from a micronuclear derivative. Macronuclear development in hypotrichous ciliates includes the formation of polytene chromosomes, degradation of these chromosomes and elimination of DNA, specific fragmentation of macronuclear DNA in short gene-sized DNA molecules and specific amplification of these molecules. After fusion of the two haplid micronuclei, the zygote nucleus divides mitotically; one of the daughter nuclei develops into a new micronucleus and the other into a new macronucleus. A first DNA synthesis phase in the developing macronucleus (macronuclear anlage) leads to the formation of polytene chromosomes. These polytene chromosomes become degraded and up to over 90% of the DNA is eliminated, leading to a DNA-poor stage. During a second DNA synthesis phase, replication bands become visible, and finally the new vegetative macronucleus is formed (for a review see Ammermann et al. 1974, Prescott 1994, Lippe & Eder 1996). As in most other ciliates analysed so far (for a review see Prescott 1994), rDNA occurs as a single-copy gene in the micronucleus but is highly amplified in the vegetative macronucleus (Steinbrück 1990). This amplification of rDNA is accompanied by the formation of many new nucleoli in the course of macronuclear development. In the light microscope, nucleoli become first visible at the beginning of the second DNA synthesis phases and multiply in subsequent rounds of replication (Ammermann et al. 1974). In this report, we describe the amplification of rDNA and the formation of new nucleoli during macronuclear differentiation by in situ hybridization of rDNA to different stages of the developing macronucleus.