Overexpression and purification of recombinant atrial natriuretic peptide using hybrid fusion protein REF-ANP in Escherichia coli.
Atrial natriuretic peptide (ANP), a small peptide consisting of 28 amino acids, has been applied in clinical treatment for heart failure, but it can encounter proteolytic degradation during its expression in host cells. Therefore, it is usually reported that ANP was expressed as a part of fusion protein. The aim of our study was to use an overexpression system to express the fusion protein REF-ANP and to optimize a purification method. First, Escherichia coli DH5alpha was transformed with constructed expression vector containing two tandem copies of ref-anp gene and the fusion protein REF-ANP was overexpressed in shaking flask culture. Subsequently, the inclusion bodies were purified with reverse phase chromatography and pooled fractions were lyophilized. After this step, REF-ANP can be solubilized under native conditions without urea. After cleavage reaction, the sample was subjected to size exclusion chromatography and then rANP was polished with reverse phase chromatography. The final purity of rANP was more than 98% and the recovery of rANP per liter of shaking flask culture was more than 3mg. Such methods as mass spectrometry, capillary isoelectrofocusing analysis, and N-terminal amino acid sequence were used to identify rANP. The capillary isoelectrofocusing analysis showed that the pI of ANP was about pH 9.7. In this study, an efficient refolding and purification process should make scaling-up procedures easier and more successful than earlier reports. Moreover, it is possible that the refolding and purification method along with the overexpression system described in this article may offer new ideas on optimizing expression and purification of other kinds of short peptides.
Wang
Jun
J
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, PR China.
Chen
Weijing
W
Lu
Jinzhi
J
Lu
Shengdong
S
D009320:Atrial Natriuretic Factor / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D002479:Inclusion Bodies / Q000737:chemistry; D017510:Protein Folding; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D009320:Atrial Natriuretic Factor