Development of a regulatable low-temperature protein expression system using the psychrotrophic bacterium, <i>Shewanella livingstonensis</i> Ac10, as the host

A low-temperature protein expression system is useful for the production of thermolabile proteins. We previously developed a system that enables constitutive protein production at low temperatures, using the psychrotrophic bacterium Shewanella livingstonensis Ac10 as the host. To increase the utility of this system, in the present study, we introduced a repressible promoter of the trp operon of this bacterium into the system. When ß-lactamase was produced under the control of this promoter at 18°C and 4°C, the yields were 75 and 33 mg/L-culture, respectively, in the absence of L-Trp, and the yields were decreased by 72% and 77%, respectively, in the presence of L-Trp. We also found that 3-indoleacrylic acid, a competitive inhibitor of the Escherichia coli trp repressor, increased the expression of the reporter gene. This repressible gene expression system would be useful for regulatable recombinant protein production at low temperatures. A regulatable low-temperature protein expression system was constructed by using a cold-adapted bacterium as the host and its trp operon promoter.</p

Additional file 3 of Division of the role and physiological impact of multiple lysophosphatidic acid acyltransferase paralogs

Additional file 3

Additional file 5 of Division of the role and physiological impact of multiple lysophosphatidic acid acyltransferase paralogs

Additional file 5

Additional file 4 of Division of the role and physiological impact of multiple lysophosphatidic acid acyltransferase paralogs

Additional file 4

Additional file 2 of Division of the role and physiological impact of multiple lysophosphatidic acid acyltransferase paralogs

Additional file 2.

Additional file 6 of Division of the role and physiological impact of multiple lysophosphatidic acid acyltransferase paralogs

Additional file 6

Additional file 1 of Division of the role and physiological impact of multiple lysophosphatidic acid acyltransferase paralogs

Additional file 1

Effects of EPA, DHA and OLA on the distribution of ANXA2, S100A10 and cavin-1 in Triton X-100-soluble (TS, 2.6% of the total volume) and insoluble (TIS, 1.3% of the total volume) fractions (A), on Tyr-phosphorylated proteins analyzed by using anti-phosphotyrosine mAb (B) and on Src that was phosphorylated either at Tyr418 and Tyr529 (C).

<p>Proteins were analyzed by immunoblotting. EPA and DHA did not affect the amounts of ANXA2, S100A10 and cavin-1 in the TIS fraction (A). The intensities of Tyr-phosphorylated protein at 36 kDa were different in the presence of EPA and DHA (B). Very small amounts of p-Src(Y418) were present only in the TS fraction. p-Src(Y529) was present in both fractions with enrichment in the TS fraction regardless of the treatments with EPA and DHA.</p

Effect of protease inhibitors on the cell-surface bound and the released ANXA2.

<p>(A) Cells were treated with none (c), AEBSF (AE, 50 µM), α-2-antiplasmin (0.33 µM; apl, 0.66 µM; aph), aprotinin (pr, 1.3 µM) and chymostatin (chy, 30 µM) for 24 h. The same portions of Hank's-EGTA extracts (upper lane) and culture supernatants after centrifugation at 2,000×g for 5 min (lower lane) were analyzed. Longer arrows indicate the position of 36 kDa whereas shorter ones indicate that of 33 kDa that was present in non-treated cells. In the presence of chymostatin, formation of 33 kDa was blocked but two new bands were present. (B) Cells were treated with Hank's-EGTA in advance and cultured in the presence of indicated inhibitors. The concentration of α-2-antiplasmin was 0.66 µM (aph). Cells were also treated with this concentration of α-2-antiplasmin and chymostatin (ch + ap). After 24 h, ANXA newly extracted in Hank's-EGTA (upper lane) and in culture supernatant (lower lane) were analyzed as in (A). Other reagents were used at the same concentration as in (A). Arrows indicate the positions of 36 kDa and 33 kDa as in (A).</p

The presence of ANXA2 on the cell surface (A), the effect of EPA and DHA on this protein (B), and the release of ANXA2-bound membranes in the culture medium (C).

<p>(A) Left; Surface-bound ANXA2 was extracted in Hank's-EGTA. One-fiftieth of the total amount was examined by immunoblotting. Two bands at 36 kDa and 33 kDa were detected. Right; serial dilution of a whole lysate of the same cell after treatment with Hank's-EGTA was analyzed. Only 36 kDa was present. The numbers denote fold-dilution of the original amount. (B) Cells were washed and incubated with a new culture medium containing EPA or DHA. At indicated time, surface-bound ANXA2 was extracted in Hank's-EGTA. While the amount in non-treated cells (upper lane) was constant, it was decreased by EPA (10 µM, middle lane) and increased by DHA (lower lane). (C) ANXA2 was not only present on the surface (c) but also in the culture supernatant (m, s, p). The same portions of Hank's-EGTA and the supernatant after centrifugation at 2,000×g for 5 min (m) and additionally at 17,000×g for 30 min (s) were analyzed. All 36 kDa and most of 33 kDa were sedimented by the last centrifugation (p, 10-fold concentrated).</p