ABSTRACT
We constructed bicistronic expression system containing AH6 promoter, 5' UTR and its fore 38 bp sequence from Corynebacterium glutamicum, followed by a conserved Shine-Dalgarno (SD) sequence for xylanase expression. The two major secretory pathways signal peptide in C. glutamicum, Tat (CgR0949) and Sec (CspB) dependent signal peptide were added before xylanase for its secretion. Fed-batch cultivation was done in a 5 L jar for high-level xylanase secretion. The enzyme properties of the purified xylanase were then studied, including the effect of temperature and pH on its activity. The xylanase could be secreted into the culture supernatant when the Sec-dependent signal peptide CspB was used, but none was detected when CgR0949 was used. The secretory production level of xylanase in a flask was 486.2 U/mL and become 1 648.7 U/mL when in a 5 L jar, which was 3.4 fold as in the flask. The optimal pH and temperature of xylanase were pH 4.5 and 45 ℃, respectively. Its activity was 80% of initial activity after pretreatment at 4 ℃ for 24 h at pH 4-11, 95% after incubation below 50 ℃ for 15 min, and 20% when the temperature above 60 ℃. The xylanase could be efficiently secreted into the culture medium by C. glutamicum using its own genetic elements, and the secretion level could be improved through large-scale fed-batch cultivation. This bicistronic expression system can provide a useful tool for heterologous proteins secretion in C. glutamicum. In addition, the catalyze activity of xylanase could be further improved by enzyme properties study.
Subject(s)
Corynebacterium glutamicum , Promoter Regions, Genetic , Protein Sorting Signals , Protein TransportABSTRACT
<p><b>OBJECTIVE</b>To explore the role of eukaryotic translation elongation factor 1A1 (eEF1A1) in regulating the invasion and metastasis of hepatocellular carcinoma (HCC) cells and the possible mechanism.</p><p><b>METHODS</b>qRT-PCR and Western blotting were used to detect the mRNA and protein expression of eEF1A1 and NOB1 in different HCC cell lines and normal liver cells. The invasion and migration abilities of HCC cells with eEF1A1 knockdown or overexpression were examined using Transwell chamber assay and RTCA assay, and the changes in NOB1 mRNA and protein expressions in the cells were detected. The effects of increasing NOB1 expression in HCCLM3-sheEF1A1 cells and decreasing NOB1 expression in eEF1A1-overexpressing MHCC97h cells on eEF1A1 expression and cell invasion and migration abilities were analyzed using Western blotting, Transwell chamber assay and RTCA assay.</p><p><b>RESULTS</b>The expressions of eEF1A1 and NOB1 were significantly increased in positive correlation in HCC cells as compared with normal hepatocytes. Knockdown of eEF1A1 significantly decreased the invasion and migration of HCC cells and reduced the mRNA and protein expression of NOB1 ( < 0.01). Overexpression of eEF1A1 significantly enhanced invasion and migration of HCC cells and increased NOB1 mRNA and protein expressions ( < 0.01). Increasing NOB1 expression in HCCLM3-sheEF1A1 cells led to the restoration of NOB1 expression and cell invasion and migration abilities ( < 0.01), whereas decreasing NOB1 in MHCC97h-eEF1A1 cells resulted in inhibition of NOB1 expression and cell invasion and migration ( < 0.01).</p><p><b>CONCLUSIONS</b>eEF1A1 positively regulates the expression of NOB1 to promote the invasion and migration of HCC cells .</p>
ABSTRACT
We constructed different N-terminal truncated variants based on Bacillus acidopullulyticus pullulanase 3D structure (PDB code 2WAN), and studied the effects of truncated mutation on soluble expression, enzymatic properties, and application in saccharification. Upon expression, the variants of X45 domain deletion existed as inclusion bodies, whereas deletion of CBM41 domain had an effective effect on soluble expression level. The variants that lack of CBM41 (M1), lack of X25 (M3), and lack both of CBM41 and X25 (M5) had the same optimal pH (5.0) and optimal temperature (60 degrees C) with the wild-type pullulanase (WT). The K(m) of M1 and M5 were 1.42 mg/mL and 1.85 mg/mL, respectively, 2.4- and 3.1-fold higher than that of the WT. k(cat)/K(m) value of M5 was 40% lower than that of the WT. Substrate specificity results show that the enzymes exhibited greater activity with the low-molecular-weight dextrin than with high-molecular-weight soluble starch. When pullulanases were added to the saccharification reaction system, the dextrose equivalent of the WT, M1, M3, and M5 were 93.6%, 94.7%, 94.5%, and93.1%, respectively. These results indicate that the deletion of CBM41 domain and/or X25 domain did not affect the practical application in starch saccharification process. Furthermore, low-molecular-weight variants facilitate the heterologous expression. Truncated variants may be more suitable for industrial production than the WT.
Subject(s)
Bacillus , Glycoside Hydrolases , Metabolism , Molecular Weight , Protein Conformation , Sequence Deletion , Substrate Specificity , TemperatureABSTRACT
Background and purpose: Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) gene is a kind of tumor suppressors, which has been reported to be underexpressed in endometrial carcinoma (EC) tissues by several reports. However, the biological effects and possible mechanisms of PTEN on EC have been known less. In this study, we tried to investigate the effects and possible mechanisms of PTEN on the invasion and migration of endometrial carcinoma cells and to provide a potential target for endometrial carcinoma therapy. Methods:The recombinant plasmid pIRES2-ZsGreen1-PTEN was rebuilt by gene recombination technology;The plasmid was transferred into HEC-1B cells and the cells transfected with pIRES2-ZsGreen1 plasmid were used as control;The expression of PTEN was observed by fluorescence microscope and Western blot assay;Cell migration and invasion was determined by the wound healing assay, transwell migration and invasion assays respectively;The Western blot analysis was performed to detect the expression of ATP-dependent tyrosine kinase (AKT), phosphorylated-AKT (p-AKT) and matrix metalloproteinase-2 (MMP-2). Results:The agarose gel electrophoresis showed a stripe of 1.2 kb which was same to PTEN cDNA;The sequence analysis showed the PCR products owned the same sequence with the coding region of PTEN cDNA in GenBank, suggesting the recombinant plasmid was constructed successfully;The green light of cells observed by fluorescence microscope and the Western blot analysis showed the expression of PTEN was upregulated in the cells transfected with the recombinant plasmid, suggesting the plasmid expressed successfully in HEC-1B cells;The wound healing assay as well as transwell migration assay showed ectopic expression of PTEN suppressed cell migration;The invasive capacity of HEC-1B cells was significantly decreased upon transfection with PTEN plasmid compared to control and untreated groups;Moreover, compared with the control groups, the expression of p-AKT and MMP-2 was downregulated, while there was no significant alteration of the expression of AKT. Conclusion:PTEN could suppress cell migratory and invasive ability of endometrial carcinoma cells by suppressing the phosphorylation of AKT followed by the decrease of MMP-2.