Construction and prokaryotic expression of SLC
- VernacularTitle:人趋化因子SLC原核表达载体的构建与表达
- Author:
Fuokang LUO
;
Hong ZHENG
;
Fengtian HE
;
- Publication Type:Journal Article
- Keywords:
secondary lymphoid tissue chemokine;
mutation;
fusion expression;
vector;
protein purification
- From:Journal of Third Military Medical University
2003;0(14):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct an expression vector pET32a(+)/SLC and express the fusion protein TrxA SLC. Methods Total RNA from human inflammatory tonsil was extracted and its cDNA was generated with reverse transcription. Mature secondary lymphoid tissue chemokine (SLC) gene was amplified by RT PCR and cloned into plasmid pET32a(+) with Nco Ⅰ and Eco RⅠ sites added to the 5′ and 3′ ends respectively. E. coli DH5? was transformed with the recombinant plasmid, and the grown clones were selected. The inserted DNA was verified by enzyme digestion and DNA sequencing. The 3 amino acids between enterokinase site and target gene were deleted with mutation and the new vector was verified by sequencing. Expression of SLC was analysed by SDS PAGE. The fusion protein was purified by metal affinity chromatography and weak cation exchange chromatography, which was analysed by SDS PAGE and Western blotting. Results Trx SLC fusion protein expression vector was successfully construced, and the fusion protein was expressed with solubility. The purified fusion protein displayed the ability of binding the goat anti human SLC polyclonal antibody. Conclusion The SLC fusion protein can be expressed with stability and solubility and primary purification is performed with metal affinity chromatography and weak cation exchange chromatography.
