Purification and characterization of the second Streptomyces phospholipase A sub(2) refolded from an inclusion body

• Published in: Protein expression and purification 2006-11, Vol.50 (1), p.82-88
• Main Authors: Jovel, Santa Romero, Kumagai, Takanori, Danshiitsoodol, Narandalai, Matoba, Yasuyuki, Nishimura, Motohiro, Sugiyama, Masanori
• Format: Article
• Language: English
• Subjects: Escherichia coli; Streptomyces; Streptomyces coelicolor; Streptomyces violaceoruber
• ISSN: 1046-5928
• DOI: 10.1016/j.pep.2006.05.009

A secreted phospholipase A sub(2) (PLA sub(2)) from Streptomyces violaceoruber A-2688, previously identified by us, is the first PLA sub(2) identified in prokaryotes. Genome sequence data of Streptomyces coelicolor A3(2) indicates that the bacterium carries two genes encoding hypothetical PLA sub(2)s, which exhibit 100 and 78% identity, respectively, to the S. violaceoruber PLA sub(2). In this study, we named the former and latter proteins as the first and second PLA sub(2)s, respectively. When the second PLA sub(2) was expressed in Escherichia coli cells, it formed an inclusion body. The present study demonstrates a method to purify it to homogeneity without the disappearance of the enzymatic activity: the inclusion body was washed with sodium deoxycholate and dissolved in the presence of 2 M urea at pH 12, then refolded by the dilution method. The refolding of enzyme was confirmed by the circular dichroism spectrum. The second PLA sub(2) purified to homogeneity had the same specific activity as that of the S. violaceoruber PLA sub(2) and the yield was approximately 6.8 mg/L culture. The second PLA sub(2) exhibits similar enzymatic properties to the S. violaceoruber PLA sub(2), except that the former enzyme does not utilize phophatidic acid as a substrate. The surface electrostatic potential of the S. coelicolor PLA sub(2) model, which is created by the computer-homology modeling, suggests that the positively charged surface of the enzyme does not affect the substrate specificity.