Efforts on directed evolution of sortase A to optimize its catalytic properties have been undertaken and shown the promise. To facilitate screening of sortase A mutants with expected catalytic properties, a novel ligation efficiency monitoring system, including reporter substrates GFP-LPETG and GGGYK-Biotin, was developed. GFP-LPETG, wild type sortase A, and a recently reported high activity sortase A mutant were prepared recombinantly from Escherichia coli BL21 (DE3). Taking advantage of the newly designed reporter system, the ligation efficiency catalyzed by wild type and mutant form of sortase A could be sensitively monitored via SDS-PAGE directly. Consistent with previous report, the mutant sortase A displayed much higher catalytic activity compared to wild type enzyme, indicating the new reporter system is easily and fast handled and sensitive. The application of this reporter system into systemic screening will facilitate future directed optimization of sortase A.
Aminoacyltransferases ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Biocatalysis ; Biotin ; Cysteine Endopeptidases ; genetics ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Genes, Reporter ; Ligation ; Mutant Proteins ; genetics ; metabolism

Objective: To analyze the expression and prognostic significance of esophageal squamous cell carcinoma associated long non-coding RNA-1 (ESCCAL-1) in esophageal squamous cell carcinoma (ESCC) tissues. Methods: From August 2011 to May 2013, 73 patients with ESCC, who received radical resection in The First Affiliated Hospital of Zhengzhou University and Henan Cancer Hospital, were enrolled. The expressions of ESCCAL-1 in esophageal tumor tissues and corresponding adjacent non-tumor tissues were detected by quantitative real-time polymerase chain reaction (qRT-PCR). T test, chi square test and multivariate analysis were performed for statistical analysis. Results: The expression of ESCCAL-1 was 28.03±9.37 in esophageal tumor tissues of patients with ESCC, which was higher than that in corresponding adjacent normal tissues (11.39±4.15), and the difference was statistically significant (t=2.964, P<0.01). However there were no statistically significant differences in the expressions of ESCCAL-1 among the patients with different age, gender, histological grade, classification of Union for International Cancer Control (UICC), T stage or lymph nodes metastasis (all P>0.05). The median disease-free survival (DFS) time and overall survival (OS) time of patients with low ESCCAL-1 expression were 39 months and 42 months, respectively, which were longer than those of patients with high ESCCAL-1 expression (30 months and 37 months), and the differences were statistically significant (χ²=9.049, P=0.003; χ²=10.165, P=0.001). The results of multivariate analysis showed that ESCCAL-1 expression was the independent risk factor of DFS and OS (high risk (HR)=2.45, 95% confidence interval (CI) 1.22 to 4.93, P=0.012; HR=2.29, 95%CI 1.14 to 4.59, P=0.019). Conclusions ESCCAL-1 may be involved the genesis and development of ESCC. The expression of ESCCAL-1 in esophageal tumor tissues may be a prognostic parameter for patients with ESCC receiving radical resection.