Isolation and purification of trans-activator of transcription -superoxide dismutase from recombinant Pichia pastoris
10.13200/j.cnki.cjb.004651
- VernacularTitle:毕赤酵母菌表达的反式转录激活因子-超氧化物歧化酶的分离纯化
- Author:
Jiansen ZHOU
1
Author Information
1. Fujian Forestry Vocational Technical College, Nanping 353000, Fujian Province, China
- Publication Type:Journal Article
- Keywords:
Superoxide dismutase(SOD);
Trans-activator of transcription(TAT);
Macroporous resin;
Pigment;
Bacterial endotoxin;
Isolation and purification
- From:
Chinese Journal of Biologicals
2026;39(02):222-228
- CountryChina
- Language:Chinese
-
Abstract:
Objective To isolate and purify the fusion protein TAT-SOD of superoxide dismutase(SOD) and the protein transduction domain of trans-activator of transcription(TAT) expressed by human immunodeficiency virus typeⅠ(HIV) using the lyophilized powder of recombinant Pichia pastoris fermentation broth as raw material.Methods The pigment and bacterial endotoxin in TAT-SOD were removed by ethanol precipitation, macroporous resin column chromatography, SP-650 ion exchange column chromatography and TritonX-114 phase separation method.Results The enzyme activity recovery rate of 70% ethanol settling TAT-SOD reached(98. 50 ± 1. 71)%, and the purification factor was(1. 04 ± 0. 02). The overall effect of separating and purifying TAT-SOD with macroporous resin was better than SP-650. The purification factor of TATSOD by macroporous resin was(3. 07 ± 0. 06) times, slightly higher than the(2. 78 ± 0. 05) times of SP-650; The removal rate of pigment in TAT-SOD by macroporous resin was(95. 16 ± 1. 65)%, which was much higher than the(44. 73 ± 0. 77)%of SP-650; The enzyme activity recovery rate of TAT-SOD by macroporous resin was(61. 17 ± 1. 06)%, slightly lower than the(69. 93 ± 1. 21)% of SP-650. The purification of TAT-SOD using Triton X-114 phase separation method resulted in a bacterial endotoxin removal rate of(99. 89 ± 1. 73)% and an enzyme activity recovery rate of(95. 02 ± 1. 66)%. The final obtained TAT-SOD had a specific activity of(10 110 ± 175) U/mg.Conclusion The combined method of ethanol precipitation, macroporous resin column chromatography, and TritonX-114 phase separation has good separation and purification effects on TAT-SOD, and is expected to provide a new option for the industrial separation and purification of TAT-SOD.
