Separation and isolation of fusion protein using a new native preparative PAGE device.
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.
College of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, People's Republic of China. biodrugcpu@yahoo.com
Zhang Xin-Guo Chen
ZX
Jian-Hua
Yan
Y
Lu-Yin
Tang
T
Li
Wang
W
Min
Cheng
C
Dai-Shuang
D000818:Animals; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods*; D004912:Erythrocytes / Q000737:chemistry; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry; D010843:Pichia / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012394:Rosaniline Dyes / Q000737:chemistry; D012397:Rosette Formation; D012709:Serum Albumin / Q000737:chemistry / Q000302:isolation & purification; D012756:Sheep; D000077596:Thymalfasin; D013947:Thymosin / Q000031:analogs & derivatives / Q000737:chemistry / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D012394:Rosaniline Dyes; D012709:Serum Albumin; C024757:Coomassie brilliant blue R; D013947:Thymosin; D000077596:Thymalfasin