Amino-Terminal Charge Affects the Periplasmic Accumulation of Recombinant Heregulin/EGF Hybrids Exported Using theEscherichia coliAlkaline Phosphatase Signal Sequence

AnEscherichia coliexpression system that exploits the bacterial alkaline phosphatase (PhoA) signal sequence to translocate recombinant human epidermal growth factor (hEGF) to the periplasm was used to evaluate how changes in the composition and sequence of amino acids near the PhoA–hEGF junction influence the periplasmic accumulation of recombinant protein. A series of chimeric structural genes was generated byin vitroreplacement of hEGF sequence with analogous segments from the EGF-like domain of human heregulin (HRG), significantly altering the electrostatic character of the amino-terminal region of the mature protein. Quantitation of HRG/EGF protein inE. coliperiplasmic extracts, by RP-HPLC, showed a fourfold decrease after one of two acidic residues located in the amino-terminal region of the mature hEGF, near the PhoA junction, was replaced. An additional threefold decrease was observed when the second acidic residue was replaced with a positively charged lysine. Further extension of the amino-terminal HRG sequence, beyond the first six residues, resulted in net neutralization of a more distant EGF acidic residue with no additional effect on protein yield. The importance of having a negatively charged group in the amino-terminal region of the mature protein was confirmed when insertion of an aspartic acid near the amino-terminus of two poorly expressed hybrid protein sequences resulted in a five- to eightfold increase in their recovery from the periplasm. This study demonstrates the importance of having negatively charged residues near the fusion junction of recombinant proteins expressed inE. coliusing the PhoA signal sequence for protein export.     

A histofluorescence study of events accompanying accumulation and migration of norepinephrine within locally cooled nerves

Glyoxylic acid was used to induce fluorescence in sections of rabbit sciatic nerve. In fresh nerves treated with this agent there were scattered finely beaded axons with a weak blue-green fluorescence. During local cooling, blue—green fluorescence accumulated steadily at the proximal boundary of the cooled region but never at its distal boundary. This accumulation gave rise to dilated axons that often swelled into brilliantly fluorescent balloon-like structures up to 10 μm in diameter. Axonal fluorescence was probably specific for norepinephrine, being enhanced by inhibition of the metabolism and diminished by inhibition of the synthesis or storage of this neurotransmitter. After local cooling of nerves for 1.5 hr, specific fluorescence was confined within 0.8 mm of the cooled region. Rewarming led to rapid removal of fluorescence from the cooled region and to disappearance of most of the balloon-like swellings. Simultaneously, rewarming caused brightly fluorescent fibers that were neither dilated nor swollen to appear in distal regions of nerve. As this wave of fluorescence migrated distally with increasing duration of rewarming, it was spread over increasingly broad regions of nerve, which suggests that axonal transport of norepinephrine may involve some kind of dispersive process.     

The role of GABA in dyskinesias induced by chemical stimulation of the striatum

The gamma aminobutyric acid (GABA) antagonists picrotoxin and bicuculline were injected into the striatum of rats via chronic cannulae. Dyskinesias were produced by these drugs which could be blocked by their injection combined with GABA. The intrastriatal (i.s.) injection of a cholinergic drug, carbachol, also produced dyskinesias which were blocked by GABA. The use of i.s. injection of GABA antagonists to produce an animal model of Huntington's chorea is discussed.     

Co-fermentation of xylose and cellobiose by an engineered Saccharomyces cerevisiae

We have integrated and coordinately expressed in Saccharomyces cerevisiae a xylose isomerase and cellobiose phosphorylase from Ruminococcus flavefaciens that enables fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. The native xylose isomerase was active in cell-free extracts from yeast transformants containing a single integrated copy of the gene. We improved the activity of the enzyme and its affinity for xylose by modifications to the 5′-end of the gene, site-directed mutagenesis, and codon optimization. The improved enzyme, designated RfCO*, demonstrated a 4.8-fold increase in activity compared to the native xylose isomerase, with a K_m for xylose of 66.7?mM and a specific activity of 1.41?μmol/min/mg. In comparison, the native xylose isomerase was found to have a K_m for xylose of 117.1?mM and a specific activity of 0.29?μmol/min/mg. The coordinate over-expression of RfCO* along with cellobiose phosphorylase, cellobiose transporters, the endogenous genes GAL2 and XKS1, and disruption of the native PHO13 and GRE3 genes allowed the fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. Interestingly, this strain was unable to utilize xylose or cellobiose as a sole carbon source for growth under anaerobic conditions, thus minimizing yield loss to biomass formation and maximizing ethanol yield during their fermentation.     

Inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase by ribulose-1,5-bisphosphate epimerization and degradation products

Xylulose-1,5-bisphosphate in preparations of ribulose-1,5-bisphosphate (ribulose-P₂) arises from non-enzymic epimerization and inhibits the enzyme. Another inhibitor, a diketo degradation product from ribulose-P₂, is also present. Both compounds simulate the substrate inhibition of ribulose-P₂ carboxylase/oxygenase previously reported for ribulose-P₂. Freshly prepared ribulose-P₂ had little inhibitory activity. The instability of ribulose-P₂ may be one reason for a high level of ribulose-P₂ carboxylase in chloroplasts where the molarity of active sites exceeds that of ribulose-P₂. Because the K_D of the enzyme/substrate complex is ≤1 μM, all ribulose-P₂ generated in situ may be stored as this complex to prevent decomposition.