ABSTRACT
Aim To obtain high pure hPPAR?1LBD fusion protein.Methods A cDNA encoding ligand binding domain(LBD)of PPAR?1 was amplified by RT-PCR from human fatty tissue and the product was inserted into the downstream of the malE gene in the vector pMAL-p2X,which encoded maltose-binding protein(MBP).The recombinant plasmid containing MBP-PPAR?1 gene was transformed into E.coli.TB1 and the expression conditions of the recombinant strain were optimized.Results The DNA strap of MW(909 bp) was presented after re-combinant plasmid was digested by Hind Ⅲ and BamH Ⅰ.The high efficient expression of MBP-PPAR?1 fusion protein in TB1 cells was observed with 38.54% product of the total cytoplasm proteins when 0.4 mmol?L-1 IPTG and 6 h incubation were taken at 30℃.Conclusion The recombinant vector was successfully constructed.It could high efficiently express hPPAR?1LBD fusion protein in TB1 cells and obtain the hPPAR?1LBD fusion protein with high bioactivity.
ABSTRACT
In order to improve the expression level of segment of GABAA receptor a1 subunit in E. coli, growth conditions of the recombatant, which influence the final yield of protein expression, including growth medium, inoculation ratio, temperature, pH, rotation speed, inducing time and concentration of IPTG and so on, were studied in shaking flasks. The results indicated that, with 3% inoculation ratio, cultured 3.5 hours at 37℃, and then induced 5 hours by IPTG at 32℃, the yield of GABAA receptor protein was 95mg/L and the biomass was 3.25 g/ L. In contrast, using a 16 L stirred fermentor instead of shaking flasks, the highest level of the protein expression, 136mg/L with 4.95g/L of biomass, was achieved after fermenting 5.5 hours.
ABSTRACT
Utilizing Polmerase Chain Reaction (PCR) and recombination DNA techniques with synthetic oligonucleotide primers,a high level expressing clone for human IL- 6, pBMhIL6, was constructed successfully in our laboratory. The intact human rIL-6 protein molecular was expressed under the control of p_Rp_L promoters, synthetic SD sequence and the terminal codon TAA which replaced TAG of original human IL-6 in E. coli. the expressed human rIL-6 protein, molecular wight 21 KDa, with specific binding to Anti-IL-6 McAb, constituted about 28% of the total cellular proteins assessed by SDS-PAGE, densitometry analysis and Western Blot assay. The results of study on kinetics of inducing human rIL-6 expression in the defferent E. coli strains and influence of bacteria growth states ingdicate that the higher productive ratio for human rIL-6 inducing expression was obtainde in E. coli DH5a at O. D=0. 7. 5?10~6 unites of human rIL-6 HPGF bioactivities were produced from 1 litre of bacteria extracts assayed by 3HTdR uptake of 7TD1 cell line.