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Production and purification of human growth-hormone-releasing factor from continuous cultures of recombinant-plasmid-containing Escherichia coli.

A recombinant gene comprising phoS (the gene for the phosphate-binding protein PhoS) fused to a synthetic gene for a modified human growth-hormone-releasing factor (mhGRF) has been constructed. This gene was highly expressed in cells growing under conditions of phosphate starvation. Various conditions of continuous culture, varying in phosphate concentrations and dilution rates, have been tested to optimize the expression of the hybrid gene product (PhoS-mhGRF). Conditions were obtained such that a large amount of the hybrid protein was no longer exported as a result of saturation of export sites, which also induce the inhibition of processing of pre-PhoE and pre-OmpA. The pre-PhoS-mhGRF, accumulated in the cell, was recovered mainly in the particulate fraction after cell fractionation. This protein was purified. Besides the methionine residues located within the signal sequence, the only other one is located in the fusion joint of the hybrid protein. Thus cyanogen bromide treatment allowed the isolation of pure mhGRF. The yield obtained is about of 1 mg/l culture.

Direct evidence for a coupling between synthesis and export of PhoS in E. coli

The accumulation of pre-PhoS under conditions of PhoS overproduction has been previously described. It is now demonstrated that during the induction of PhoS, a delay in the completion of polypeptide chain elongation can be detected. This delay is related to the extent of jamming of export sites by pre-PhoS or by other exported proteins. These results suggest that a component required for completion of pre-PhoS polypeptide becomes limiting, being titrated by the excess of nascent chains bearing signal peptides. This component thus probably acts at an early step in the export pathway.

Protein export in Escherichia coli

Hyperproduction of phosphate-binding protein (PhoS) resulted in saturation of export sites, and pre-PhoS was accumulated both in the inner membrane and in the cytoplasm. The cytoplasmic pre-PhoS could not be exported post-translationally; only the membrane-associated precursor could be matured and exported. Preliminary evidence has been obtained for the existence of a translation stop. The pause site in pre-PhoS mRNA translation occurs when 70 to 80 amino acids have been assembled, and appears to be related to the coupling of synthesis and export.

Normal precursors of periplasmic proteins accumulated in the cytoplasm are not exported post-translationally in Escherichia coli.

Hyperproduction of phosphate-binding protein, PhoS, in strains carrying a multicopy plasmic containing the phoS gene, resulted in saturation of export sites. As a consequence, pre-PhoS was accumulated both in the inner membrane and in the cytoplasm. This was evidenced both in electron-microscopy and after cell fractionation. Only the membrane-associated precursor could be matured and exported. The signal sequence of the cytoplasmic pre-PhoS was slowly degraded. It was first cleaved about in its middle and then completely removed. Electron microscope studies demonstrated that the cytoplasmic pre-PhoS cannot be exported post-translationally.

Purification and characterization of pro-TraTp, the signal sequence-containing precursor of a secreted protein encoded by the F sex factor.

The structural gene for the F sex factor outer membrane surface exclusion protein ( traT ) was cloned onto a high-copy-number plasmid where it is expressed from the phage lambda promoter pL. Conditional control over expression was provided by a temperature-sensitive lambda cI repressor. Induction of pL produced large quantities of the traT gene product ( TraTp ) and, in rich growth media, even larger amounts of a higher-molecular-weight form of TraTp . This polypeptide was purified and characterized as a pro- TraTp precursor, which contains at its amino terminus a typical signal-like sequence, which is not present in the mature form of TraTp as isolated from the outer membrane of F-containing cells. Accumulation of pro- TraTp seemed not to result from the jamming of export sites, as in another system for obtaining precursors of secreted proteins, but rather from overwhelming kinetically the ability of the cell to process exported proteins. Although pro- TraTp appeared to be successfully translocated to the outer membrane, it was defective in forming the oligomeric structure required for surface exclusion function. The procedure used is not a general method but can be applied to certain other secreted proteins.

Gene fusions using the ompA gene coding for a major outer-membrane protein of Escherichia coli K12.

It has been shown previously that fragments of the Escherichia coli major outer membrane protein OmpA lacking CO2H-terminal parts can be incorporated into this membrane in vivo [Bremer et al. (1982) Eur. J. Biochem. 122, 223-231]. The possibility that these fragments can be used, via gene fusions, as vehicles to transport other proteins to the outer membrane has been investigated. To test whether fragments of a certain size were optimal for this purpose a set of plasmids was prepared encoding 160, 193, 228, 274, and 280 NH2-terminal amino acids of the 325-residue OmpA protein. The 160-residue fragment was not assembled into the outer membrane whereas the others were all incorporated with equal efficiencies. Thus, if any kind of OmpA-associated stop transfer is required during export the corresponding signal might be present between residues 160 and 193 but not CO2H-terminal to 193. The ompA gene was fused to the gene (tet) specifying tetracycline resistance and the gene for the major antigen (vp1) of foot-and-mouth disease virus. In the former case a 584-residue chimeric protein is encoded consisting NH2-terminally of 228 OmpA residues followed by 356 CO2H-terminal residues of the 396-residue 'tetracycline resistance protein'. In the other case the same part of OmpA is followed by 250 CO2H-terminal residues of the 213-residue Vp1 plus 107 residues partly derived from another viral protein and from the vector. Full expression of both hybrids proved to be lethal. Lipophilic sequences bordered by basic residues, present in the non-OmpA parts of both hybrids were considered as candidates for the lethal effect. A plasmid was constructed which codes for 280 OmpA residues followed by a 31-residue tail containing the sequence: -Phe-Val-Ile-Met-Val-Ile-Ala-Val-Ser-Cys-Lys-. Expression of this hybrid gene was lethal but by changing the reading frame for the tail to encode another, 30-residue sequence the deleterious effect was abolished. It is possible that the sequence incriminated acts as a stop signal for transfer through the plasma membrane thereby jamming export sites for other proteins and causing lethality. If so, OmpA appears to cross the plasma membrane completely during export.

Peripheral nervous system myelin assembly in vitro: perturbation by the ionophore monensin.

Abstract: Sciatic nerves from 13‐day‐old rats were incubated in vitro with [35S]methionine in the presence or absence of 0.22 μM monensin and total paniculate and myelin fractions prepared. The total particulate was further subfractionated by continuous density gradient centrifugation, after which the maximal specific activities of three marker enzymes, 2′,3′‐cyclic nucleotide phospho‐diesterase (myelin), 5′‐nucleotidase (plasma membrane), and cerebroside sulphotransferase were recovered at 0.72, 0.82, and 0.92 M sucrose, respectively. The radiolabelled proteins present in the gradient subtractions were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) and fluorography, and bands corresponding to the P0 and myelin basic proteins were identified by co‐migration with unlabelled myelin marker proteins on both one‐dimensional SDS‐PAGE and two‐dimensional nonequilibrium isoelectric focussing/SDS‐PAGE systems. Following a 90‐min incubation with [35S]methionine, newly synthesized myelin basic proteins were recovered in fractions between 0.5 and 0.7 M sucrose; this distribution was unaltered by monensin. In contrast, the distribution of newly synthesized P0 protein across the gradients was influenced by monensin: a bimodal distribution across the control gradients with peaks of recovery of 0.60 and 0.82 M sucrose was altered to give a single peak at an intermediate density of 0.72 M sucrose. The total proportions of newly synthesized P0 and myelin basic proteins (MBP) present across the entire gradients were calculated from the fluorograms, and the ratio was found to be 2.8 P0: (LBP + SBP), in both the presence and absence of the ionophore. However, only 70% and 50% of the control levels of MBP and P0 were recovered with a purified myelin fraction after incubation with monensin. The results are discussed with reference to different intracellular transport processes for the P0 glycoprotein and the MBP within the Schwann cell, and also to the differential compartmentation of the sites of synthesis and membrane export within the Golgi body.

Evidence for a coupling of synthesis and export of an outer membrane protein in Escherichia coli.

We describe a lesion, lamB701-708, affecting the hydrophilic portion of the lambda receptor signal sequence. The C to A transversion of the sixth codon of the signal sequence changes a positively charged arginine to a neutral serine. The phenotype conferred by this alteration is unique among previously described signal sequence mutations. The results suggest an essential role for the charged amino acids of the hydrophilic segment in the initial interaction between a nascent secreted protein and a membrane export site. The results further suggest that synthesis of lambda receptor is coupled to its export.

Studies on the expression of outer membrane protein 2 in escherichia coli.

The relative level of protein 2 expressed in the outer membrane of strains of Escherichia coli K-12 lysogenized with bacteriophage PA-2 was found to be influenced by both the growth temperature and lc+ gene dosage. An increase in either of these parameters was accompanied by an increase in the level of protein 2 up to an apparent saturation level. Any increase in the amount of protein 2 was accompanied by a concomittant decrease in the amount of OmpF and OmpC porins. This inverse relationship led to the maintenance of an approximately constant protein mass per unit of peptidoglycan. Our results are discussed in light of recent genetic studies on the regulation of the OmpF and OmpC porins and can be explained through the competition of these three matrix proteins for a common export or insertion site.

Insertion of a MalE beta-galactosidase fusion protein into the envelope of Escherichia coli disrupts biogenesis of outer membrane proteins and processing of inner membrane proteins.

The synthesis of a membrane-bound MalE beta-galactosidase hybrid protein, when induced by growth of Escherichia coli on maltose, leads to inhibition of cell division and eventually a reduced rate of mass increase. In addition, the relative rate of synthesis of outer membrane proteins, but not that of inner membrane proteins, was reduced by about 50%. Kinetic experiments demonstrated that this reduction coincided with the period of maximum synthesis of the hybrid protein (and another maltose-inducible protein, LamB). The accumulation of this abnormal protein in the envelope therefore appeared specifically to inhibit the synthesis, the assembly of outer membrane proteins, or both, indicating that the hybrid protein blocks some export site or causes the sequestration of some limiting factor(s) involved in the export process. Since the MalE protein is normally located in the periplasm, the results also suggest that the synthesis of periplasmic and outer membrane proteins may involve some steps in common. The reduced rate of synthesis of outer membrane proteins was also accompanied by the accumulation in the envelope of at least one outer membrane protein and at least two inner membrane proteins as higher-molecular-weight forms, indicating that processing (removal of the N-terminal signal sequence) was also disrupted by the presence of the hybrid protein. These results may indicate that the assembly of these membrane proteins is blocked at a relatively late step rather than at the level of primary recognition of some site by the signal sequence. In addition, the results suggest that some step common to the biogenesis of quite different kinds of envelope protein is blocked by the presence of the hybrid protein.

Anionic sites on the envelope ofSalmonella typhimurium mapped with cationized ferritin

Binding of either ferritin (F) or cationized ferritin (CF) was employed to indicate the surface charge of the envelope of mainly two Salmonella typhimurium strains (395 MR10, a Rd-mutant, and LT2-M1, a UDP-galactose-4-epimerase-less mutant). Lowering the pH from 7 to 4 decreased binding of CF, but increased binding of F. At low concentrations, the distribution of CF on S. typhimurium 395 MR10 was in general random, with individual ferritin molecules often forming clusters of two or three particles. At ionic strengths of 0.25M NaCl, ferritin produced distinctive, larger clusters at relatively few sites (10-50/cell). Addition of galactose to cultures of growing S. typhimurium, LT2-M1 reduced the binding of CF in 1-10 min, and numerous ferritin-free areas became visible. Possibly this is caused by a pluri-focal reduction in the negative cell surface charge that was generated at the multiple sites of export of new, smooth-type lipopolysaccharide, which either exhibits lesser charge or masks a preexisting surface charge. Dividing cells may show unequal charges on the prospective daughter cells, and the difference in the capacity for ferritin adsorption of both daughter cells is sharply separated at the division site.

Visualization of pores (export sites) correlated with cellulose production in the envelope of the gram-negative bacterium Acetobacter xylinum.

The Gram-negative bacterium Acetobacter xylinum assembles a cellulse ribbon composed of a number of microfibrils in the longitudinal axis of its envelope. The zone of ribbon assembly was investigated by freeze-etch electron microscopy. Freeze-etching revealed, beneath the cellulose ribbons, a linear array of pores on the lipopolysaccharide membrane. These pores have a rim diameter of 120--150 A and a central hole or deepening of approximately 35 A. The axes of pore arrays closely coincide with linear arrays of 100 A particles on the E- and P-faces of the fractured lipopolysaccharide membranes. Pores and particles in the lipopolysaccharide membrane are probably congruent. The pores are hypothesized to be the export sites (penetration sites) for cellulose.

Lateral mobility and surface density of lipopolysaccharide in the outer membrane of Salmonella typhimurium.

Salmonella typhimurium 1195 mutant bacteria were allowed to produce clusters of lipopolysaccharide with new, wild‐type specificity over the export sites. Ferritin‐labeling experiments showed that the lipopolysaccharide diffuses away from the export sites at 37 °C and is found dispersed on the cell surface, whereas at 0 °C it remains immobile.The density of lipopolysaccharide molecules on the bacterial surface, measured as number of lipopolysaccharide molecules per μm2, or as percentage of the bacterial surface covered by lipopolysaccharide was determined in two wild‐type S. typhimurium strains and one mutant strain, which was grown under conditions either permissive or nonpermissive for the formation of wild‐type lipopolysaccharide. There are 0.7—1.0 × 105 lipopolysaccharide molecules per μm2 surface area. About 15—25% of the bacterial surface was found to be covered by lipopolysaccharide. The mean intermolecular distance at hypothetical uniform distribution of lipopolysaccharide was calculated to be in the range of 3—4 nm. It was of no consequence to the lipopolysaccharide surface density whether the mutant strain produced wildtype or mutant lipopolysaccharide.An estimate of the lateral diffusion constant D (D∼ 3 × 10−13 cm2/s) of lipopolysaccharide in the outer membrane was made from earlier published data [2]. Possible reasons for the low order of magnitude of D, based on the observed surface density, are discussed.

Outer membrane of salmonella. Sites of export of newly synthesised lipopolysaccharide on the bacterial surface.

The UDP‐galactose‐4‐epimeraseless strain 1195 of Salmonella typhimurium bears incomplete lipopolysaccharide, and only after addition of galactose to the growth medium it produces wild‐type lipopolysaccharide. It was demonstrated in a double‐label experiment that already existent, incomplete lipopolysaccharide is not completed after galactose addition but that only the newly synthesised lipopolysaccharide carries the wild‐type specificity.It was thus possible to locate newly synthesised lipopolysaccharide with specific anti‐wild‐type, ferritin‐conjugated antibodies in freeze‐etch preparations and ultra‐thin sections.With the freeze‐etch technique new lipopolysaccharide was found to appear in about 220 patches per cell, 30 s after 50 mM galactose had been added to the medium. After growth for 2–3 min under these conditions new lipopolysaccharide was found to be evenly spread over the entire cell surface.In ultra‐thin sections of plasmolysed cells 86% of the ferritin patches, indicating newly synthesised lipopolysaccharide, was found located over sites where the cytoplasmic and the outer membrane adhere to one another. It is discussed that new lipopolysaccharide emerges from these sites and that lateral movement of lipopolysaccharide in the outer membrane results in an even distribution on the bacterial cell surface.