Twin-arginine signal peptide of Bacillus licheniformis GlmU efficiently mediated secretory expression of protein glutaminase

Background: Protein glutaminase specifically deamidates glutamine residue in protein and therefore significantly improves protein solubility and colloidal stability of protein solution. In order to improve its preparation efficiency, we exploited the possibility for its secretory expression mediated by twin-arginine translocation (Tat) pathway in Bacillus licheniformis. Results: The B. licheniformis genome-wide twin-arginine signal peptides were analyzed. Of which, eleven candidates were cloned for construction of expression vectors to mediate the expression of Chryseobacterium proteolyticum protein glutaminase (PGA). The signal peptide of GlmU was confirmed that it significantly mediated PGA secretion into media with the maximum activity of 0.16 U/ml in Bacillus subtilis WB600. A mutant GlmU-R, being replaced the third residue aspartic acid of GlmU twin-arginine signal peptide with arginine by site-directed mutagenesis, mediated the improved secretion of PGA with about 40% increased (0.23 U/ml). In B. licheniformis CBBD302, GlmU-R mediated PGA expression in active form with the maximum yield of 6.8 U/ml in a 25-l bioreactor. Conclusions: PGA can be produced and secreted efficiently in active form via Tat pathway of B. licheniformis, an alternative expression system for the industrial-scale production of PGA.

Low expression of MRP1/GS-X pump ATPase in lymphocytes of Walker 256 tumour-bearing rats is associated with cyclopentenone prostaglandin accumulation and cancer immunodeficiency.

Immunosuppression is a life-threatening complication of late cancer stages. In this regard, overproduction in the host plasma of the anti-inflammatory cyclopentenone prostaglandins (CP-PGs), which are strongly antiproliferative at high concentrations, may impair immune function. In fact, lymphoid tissues of tumour-bearing rats accumulated large amounts of CP-PGs while the tumour tissue itself did not. Expression of the CP-PG-induced 72-kDa heat shock protein (hsp70) was elevated in lymphocytes from tumour-bearing animals related to controls. As the capacity for CP-PG uptake by lymphocytes is the same as tumour cells, we investigated whether the latter could overexpress the multidrug resistance-associated protein (MRP1/GS-X pump) which extrudes CP-PGs towards the extracellular space as glutathione S-conjugates. Walker 256 tumour cells extruded 15-fold more S-conjugates than lymphocytes from the same rats (p < 0.001). This did not appear to be related to deficiency in lymphocyte glutathione (GSH) metabolism, since the major GSH metabolic routes are consistent with CP-PG conjugation in lymphocytes. This was not the case, however, for the MRP1/GS-X pump activity in lymphocyte membranes (in pmol/min/mg protein: 3.1 +/- 1.7 from normal rats, 0.2 +/- 0.2 from tumour-bearing animals vs 64.3 +/- 7.0 in tumour cells) which was confirmed by Western blot analysis for MRP1 protein. Transfection of lymphocytes with MRP1 gene completely abolished CP-PG (0-40 microM) toxicity. Taken together, these findings suggest that CP-PG accumulation in lymphocytes may be, at least partially, responsible for cancer immunodeficiency. Clinical approaches for overexpressing MRP1/GS-X pump in lymphocytes could then play a role as a tool for the management of cancer therapeutics.