Domain fusion is limited at enzyme one terminus. The issue was explored by swapping a mesophilic Aspergillus niger GH11 xylanase (Xyn) with a hyper-thermophilic Thermotoga maritima glucanase (Glu) to construct two chimeras, Xyn-Glu and Glu-Xyn, with an intention to create thermostable xylanase containing glucanase activity.

The sequence of polypeptide N-acetylgalactosaminyltransferase 14(GALNT14) was analyzed using bioinformatics.A peptide sequence was selected according to hydrophilicity,antigenicity,flexibility and accessibility.The cDNA of interest was subcloned into a eukaryotic expression vector pET-DsbA to construct pET-DsbA-GALNT14.The expression of recombinant fusion protein was induced by IPTG,then purified by Ni-IDA Resin.Polyclonal antibody against GALNT14 was prepared from immuned New Zealand white rabbit.The titer and specificity of the prepared antibody were analyzed by immunodiffusion and Western blot.The result showed that the fusion protein was expressed successfully,and the interest protein with high purity was obtained.The generated antibody was specific.Both of MCF-7 and 786-O cell expressed GALNT14.A good foundation for further study on the function of GALNT14 and its role of tumorigenesis and development was laid.

N-acetylgalactosaminyltransferases (GalNAc-Tases) catalyze the first step in the O-glycosylation of mucin proteins.GalNAc-T14 is a new member of glycosyltransferases family.Recent studies have demonstrated that O-glycosylation may be closely related to the carcinogenesis and development of tumours.Hence,studies on the N-acetylgalactosaminyltransferase family are getting more and more attention.The nomenclature,structure,distribution,function and the potential application value of GalNAc-T14 was reviewed in this paper.

Background: The post-translational modification of proteins, including glycosylation, differs between normal and tumor cells. The UDP-N-acetyl-D-galactosamine polypeptide N-acetylgalactosaminyltransferases (GalNAc-Tases) family of enzymes regulates the initial steps of mucin O-glycosylation and is responsible for the altered glycosylation state observed in cancer cells. Recently it was found that GalNAc-T14 mRNA is heterogeneously expressed in breast carcinomas compared to normal tissue, however the expression profile of GalNAc-T14 protein in breast carcinomas compared to normal tissue is still unknown. In this study, we assessed the expression profile of GalNAc-T14 protein in malignant and non-malignant breast tissues by immunohistochemistry to evaluate whether GalNAc-T14 might be a potential biomarker for breast cancer.Methods: In formalin-fixed tissues, the expression level of GalNAc-T14 protein was evaluated by immunohistochemistry assay in breast tissues. Expression profiles were assessed in normal tissues, benign fibroadenomas and several types of carcinomas.Results: Our results showed that GalNAc-T14 was heterogeneously expressed in breast carcinomas compared to nonmalignant tissue. GalNAc-T14 expression was observed in 47/56 (83.9%) carcinoma samples, 7/48 (14.6%) nonmalignant breast tissue samples. GalNAc-T14 expression level was associated with histological grade. For this enzyme a significant association with invasive ductal type, mucinous adenocarcinoma and ductal carcinoma in situ (DCIS) type was found.Conclusion: Our results provide evidence that GalNAc-T14 may be a potential biomarker for breast cancer by immunohistochemistry. GalNAc-T14 expression level was associated with histological grade. GalNAc-T14 expression can provide new insights about breast cancer glycobiology.

In enzyme engineering,screening for transformant was labor-intensive and time-consuming.Toselect transformant containing accurate gene efficiently,we introduced an activity-screening method.Xylanasegene was cloned into pET20b and was used to transform E.coli strain BL21(DE3) competent cells.10transformants were inoculated in 0.5 mL LB for culturing overnight,9.5 mL LB was re-added and cultured for2.5 h.After induction for 5 h,cells were harvested and lyzed by freezing-thawing procedure in a simplifiedlysis buffer.For having activities closer to or higher than that of the positive control,7 transformants wereregarded as positive,thereafter,plasmid DNA was sequenced to confirm gene's accuracy.The procedurelasted for 2 to 3 days,having characteristic of high-throughput for assaying transformant's enzyme activity inparallel.Differing from screening transformant for gene of previous method,the present method screenedtransformant for enzyme activity,which eliminated those nonsense mutations.The preliminarily assayedactivities were compared for finding out transformant having high activity.

Studies were conducted on the effect of plasmid size on the over-expression of xylanase by comparing pET21a(5 443 bp) with pET20b(3 716 bp) recombinant with Aspergillus niger xylanase,which was expressed in E.coli BL21(DE3) strain.The big-sized and the small-sized plasmid cell had plasmid DNA concentration as(4.6±0.01),(5.5±0.01) g·L-1,xylanase protein concentration as(57.2±10.6),(140.2±22.1) mg·L-1,and xylanase activity as(6.6±0.2),(69.6±11.2) U·mL-1 respectively.Compared with that of the big-sized cell,each parameter of the small-sized cell increased by 19.5%,1.45-fold and 9.5-fold;indicating that size effect on xylanase expression had an increasing trend from plasmid concentration to xylanase activity.The study demonstrated that plasmid-size was important for over-expression of protein,thus,a small-sized plasmid was better for genetic engineering.