ABSTRACT
A human serum albumin and Thymosin α1 (HSA-Tα1) fusion protein was designed and over-expressed in Pichia pastoris. To purify the fusion protein, a new native preparative electrophoresis system that involved a modified device with a sample receiving chamber, and an assay method with Coomassie Blue G-250 tracing the collection of the protein of interest. In this device, two gels were run in parallel: native vertical collecting polyacrylamide gel electrophoresis (PAGE) and native vertical tracing PAGE. Samples mixed with or without Coomassie Blue G-250 loading buffer were separately loaded to the two aforementioned gels, and the fractions were collected until the tracing protein band combined with dye reached 1 cm from the sample-receiving chamber at the bottom of the gel. Approximately nine fractions were collected at regular intervals of 15 min. HSA-Tα1 fusion protein with 95% relative homogeneity was harvested and manifested similar immunological activities as synthetic Tα1 after a single-step purification of this preparative PAGE. As a result, this system offers a new, rapid and simple method for the purification of the protein of interest.
Subject(s)
Electrophoresis, Polyacrylamide Gel/methods , Recombinant Fusion Proteins/isolation & purification , Animals , Cells, Cultured , Electrophoresis, Polyacrylamide Gel/instrumentation , Erythrocytes/chemistry , Humans , Lymphocytes/chemistry , Pichia/chemistry , Recombinant Fusion Proteins/chemistry , Rosaniline Dyes/chemistry , Rosette Formation , Serum Albumin/chemistry , Serum Albumin/isolation & purification , Sheep , Thymalfasin , Thymosin/analogs & derivatives , Thymosin/chemistry , Thymosin/isolation & purificationABSTRACT
OBJECTIVE: To study the effect of ursolic acid (UA), apentacyclic triterpene acid, on MCF-7 cell apoptosis, and probable mechanism involved by detecting the expressions of caspase-3 and poly ADP-ribose polymerase(PARP) at protein level. METHOD: MCF-7 cells were cultured with different concentrations of UA. Growth inhibition of UA on MCF-7 cells was evaluated by MTT assay. Cell cycle and sub-G1 peak were performed by FCM. Morphologic changes of UA-treated cells were observed by light microscope. Apoptotic cells with condensed or fragmented nuclei were visualized by Ho 33258 staining by a fluorescence microscope (EX: U. V.). The protein expression of caspase-3 and PARP was analyzed by immunofluorescence cell staining (SABC-Cy3). RESULT: 24 hours after UA treatment, inhibition of MCF-7 cell growth was concentration-dependent. The IC50 value for UA was (22.6 +/- 3.0) micromo x L(-1). Cell cycle anaysis by FCM showed that 50 micromol x L(-1) of UA arrested MCF-7 cell cycle at G0 - G1 phase. Morphological changes of MCF-7 Cells exhibited many of the hallmark features of apoptosis, including chromatin clumps and aggregation and DNA fragmentation. UA increased caspase-3 protein expression. CONCLUSION: The results suggest that UA evokes MCF-7 cell apoptosis is correlation with the up-regulation of caspase-3. Our study indicated that UA might be a potential Chinese medical component for breast neoplasm.
