ABSTRACT
Arabinose-5-phosphate isomerase (KdsD) is the first key limiting enzyme in the biosynthesis of 3-deoxy-D-manno-octulosonate (KDO). KdsD gene was cloned into prokaryotic expression vector pET-HTT by seamless DNA cloning method and the amount of soluble recombinant protein was expressed in a soluble form in E. coli BL21 (DE3) after induction of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The target protein was separated and purified by Ni-NTA affinity chromatography and size exclusion chromatography, and its purity was more than 85%. Size exclusion chromatography showed that KdsD protein existed in three forms: polymers, dimmers, and monomers in water solution, different from microbial KdsD enzyme with the four polymers in water solution. Further, the purified protein was identified through Western blotting and MALDI-TOF MASS technology. The results of activity assay showed that the optimum pH and temperature of AtKdsD isomerase activities were 8.0 and 37 ℃, respectively. The enzyme was activated by metal protease inhibitor EDTA (5 mmol/L) and inhibited by some metal ions at lower concentration, especially with Co²⁺ and Cd²⁺ metal ion. Furthermore, when D-arabinose-5-phosphate (A5P) was used as substrate, Km and Vmax of AtKdsD values were 0.16 mmol/L, 0.18 mmol/L·min. The affinity of AtKdsD was higher than KdsD in E. coli combined with substrate. Above results have laid a foundation for the KdsD protein structure and function for its potential industrial application.
Subject(s)
Aldose-Ketose Isomerases , Arabidopsis , Arabidopsis Proteins , Cloning, Molecular , Escherichia coli , Metabolism , Metals , Pentosephosphates , Recombinant ProteinsABSTRACT
Objective:To investigate the effects of epidermal growth factor (EGF)on cell cycle and cell cycle-related regulatory factors of human esophageal squamous cell carcinoma (ESCC) cell line Eca109.Methods: Serum starved Eca109 cells were treated with 20 ng/ml recombinant human EGF(rhEGF)for 24 h.The cell cycle phase distribution was detected by flow cytometry.The mRNA and protein expression levels of p21CIP1/WAF1(p21) and p27KIP1(p27) were detected by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)and Western blot,respectively.Results: The proportions of G1 phase cells in EGF group and control group were ( 54.90 ±0.82 )% and ( 65.94 ±0.74 )%.The mRNA and protein expression levels of p 21 in EGF group was significantly higher ,and p27 was significantly lower than that in control group ( P<0.01 ) .Conclusion: EGF facilitates G1-S phase transition,and promotes the proliferation of Eca 109 cells,which may be associated with the up-regulation of p21 and down-regulation of p27.
ABSTRACT
Objective To investigate the expression of Nek8 in esophageal squamous cell carcinoma(ESCC) tissues and cell line , and to evaluate its correlation with the clinicopathological features of ESCC and the survival rate of ESCC patients after operation . Methods The expression of Nek8 mRNA in human ESCC Eca109 cell line and two pairs of ESCC tissues and adjacent normal e‐sophageal mucosal epithelium were detected by semi‐quantitative reverse transcription polymerase chain reaction (RT‐PCR) .Immu‐nohistochemistry and tissue microarray technique were used to examine the expression of Nek 8 protein in ESCC tissues and tumor‐adjacent tissues .The correlation between Nek8 expression and clinicopathological features of ESCC and survival rate of ESCC pa‐tients was then analyzed .Results The expression of Nek8 mRNA was positive in Eca109 cells and two cases of ESCC tissue ,and it was negative in paired normal esophageal mucosal epithelium specimens .In tissue microarray ,the expression of Nek8 protein in ES‐CC tissues ,which was mainly in the cytoplasm ,was significantly higher than that in tumor‐adjacent tissues(P= 2 .16E‐13) .The high expression of Nek8 was associated with tumor size (P=0 .008) ,but not with sex ,age ,histological grade ,infiltration degree , lymph node metastasis ,and the survival rate(P>0 .05) .Conclusion The expression of Nek8 is up‐regulated in ESCC tissues and cell line ,and may be involved in tumorigenesis and development of ESCC .Nek8 could act as a potential biomarker for ESCC diag‐nose and target for therapy .