ABSTRACT
Objective To compare the two kinds of purification method for purifying recombinant human cardiac troponin I(cT-nI)to obtain the stable cTnI and promote the study of cTnI diagnosis standardization.Methods The cTnI inclusion body was ob-tained by the ultrasonic broken engineering,after washing by 2% Tritonx-100,2M urea,dissolved in 8M urea,then purified by the column refolding on CM-FF and the dilution refolding respectively.The cTnI yields were compared between the two kinds of meth-od and the stability at 4 ℃,20 ℃,-80 ℃ and on the freeze-dried condition was compared.Then the purification method to effi-ciently obtain the stable cTnI was established.Results The protein about 2 mg and 1.4 mg could be obtained by CM-FF on the col-umn refolding and the dilution refolding from 0.1 g of wet inclusion body,respectively.The former method had the short cycle and high efficiency.The cTnI purified by the column refolding on CM-FF was more stable at 4 ℃,20 ℃,-80 ℃ and on the freeze-dried condition.Conclusion The column refolding on CM-FF is more stable and highly efficient in purification of cTnI than the dilution refolding.
ABSTRACT
The chicken-type lysozyme of the insect Spodoptera litura (SLLyz) is a polypeptide of 121 amino acids containing four disulfide bridges and 17 rare codons and participates in innate defense as an anti-bacterial enzyme. The recombinant S. litura lysozyme (rSLLyz) expressed as a C-terminal fusion protein with glutathione S-transferase (GST) in Rosetta(DE3) Singles. The protein was produced as an inclusion body which was solubilized in 8 M urea, renatured by on-column refolding, and purified by reversed-phase chromatography to 95 percent purity. The purified rSLLyz demonstrated antibacterial activity against B. megaterium confirmed by inhibition zone assay. The overexpression and refolding strategy described in this study will provide a reliable technique for maximizing production and purification of proteins expressed as inclusion bodies in E. coli.
Subject(s)
Inclusion Bodies/metabolism , Muramidase/metabolism , Spodoptera , Anti-Bacterial Agents , Bacillus megaterium , Blotting, Western , Chromatography, Reverse-Phase , Electrophoresis , Escherichia coli , Glutathione Transferase , Protein Folding , Recombinant ProteinsABSTRACT
EC-SOD inclusion bodies was refolded on column using metal (Ni) affinity chromatography, based on the metal-binding property of His-tag. The effect of protein amount, urea removal speed and temperature on refolding was observed. We compared the different efficiency purified with Ni-sepharose and Heprain-sepharose affinity chromatography, and studied the stability of the refolded proteins. The results indicate that the inclusion bodies can be renatured with Ni-sepharose affinity chromatography. The increase of the protein amount and urea removal rate , the lower of the renaturation efficiency. Higher temperature was benefit to protein renaturation. Both the Ni-sepharose and Heprain-sepharose affinity column can be used to purified the refolded proteins, but purified by Heprain-sepharose affinity column the protein had higher activity. The activity of renatured protein was stable in 10 ℃～50℃,when pH10 its stability was lower significantly. In denaturating solution the stability of renatured protein was low.