Prokaryotic expression and functional identification of androgen receptor functional domains
10.19405/j.cnki.issn1000-1492.2023.02.008
- Author:
Yuqi Zhang
1
;
Mingzhu Ding
1
;
Jinfeng Wu
1
;
Haixiang Zheng
1
;
Lei Zhang
1
;
Xing Wang
1
Author Information
1. School of Basic Medical Sciences，Fujian Medical University，Fuzhou 350122
- Publication Type:Journal Article
- Keywords:
androgen receptor;
prokaryotic expression;
GST fusion protein;
functional domain mutants
- From:
Acta Universitatis Medicinalis Anhui
2023;58(2):219-224
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct the full-length prokaryotic expression plasmid of the wild type of androgen receptor (AR) and the truncated body of four functional domains，and to identify the fusion protein by Western blot and electrophoretic mobility shift assay ( EMSA) ．
Methods:Based on the pGEX-4T-1 vector ，the recombinant plasmids were constructed to express the full-length and functional domains of AR. IPTG was used to induce the expression of the recombinant proteins，which were isolated and purified by glutathione sepharose 4B beads under the optimized condition．The specific protein expression in the bacterial lysate and the purified protein isolated with glutathione sepharose 4B beads was identified by Western blot with AR antibody and GST labeled antibody．The purified protein was incubated with a fluorescent probe of the virus，and the complex was detected by electrophoresis in a non-denaturing gel.
Results : The prokaryotic recombinant plasmids of full length and three functional domain truncated AR were successfully constructed．The recombinant clones were identified by using bacterial culture as a template，and further verified by double enzyme digestion．It showed that there were identical bands in the same sizes as the inserted fragments．The nucleotide and the amino acid sequences were aligned to the reference sequence in NCBI GenBank．The GST fusion protein，GST-AR-NTD + DBD (96 ku) and GST-AR-NTD (86 ku) were successfully induced and verified． The purified protein could be directly combined with the viral genome DNA．
Conclusion:The prokaryotic expression conditions of truncated AR plasmid from the same gene sequence are different．The purified AR protein can be used to understand the direct interaction mechanism between functional domains of AR and other molecules.
- Full text:2024071323012521477雄激素受体基因各功能域片段的原核表达与功能鉴定_张雨琪.pdf
