Recombinant expression of bioactive peptide lunasin in Escherichia coli.
Lunasin, a cancer-preventive peptide, was isolated from soybean, barley, and wheat. Previous studies showed that this 43-amino acid peptide has the ability to suppress chemical carcinogen-induced transformation in mammalian cells and skin carcinogenesis in mice. In this study, we attempted to use the Escherichia coli T7 expression system for expression of lunasin. The lunasin gene was synthesized by overlapping extension polymerase chain reaction and expressed in E. coli BL21(DE3) with the use of vector pET29a. The recombinant lunasin containing his-tag at the C-terminus was expressed in soluble form which could be purified by immobilized metal affinity chromatography. After 4 h, the expression level is above 4.73 mg of recombinant his-tagged lunasin/L of Luria-Bertani broth. It does not affect the bacterial growth and expression levels. This is the first study that successfully uses E. coli as a host to produce valuable bioactive lunasin. The result of in vitro bioassay showed that the purified recombinant lunasin can inhibit histone acetylation. Recombinant lunasin also inhibits the release of pro-inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, and nitric oxide production). Compared with other research methods on extraction or chemical synthesis to produce lunasin, our method is very efficient in saving time and cost. In the future, it could be applied in medicine and structure-function determination.
Liu
Chin-Feng
CF
Institute of Microbiology and Biochemistry, College of Life Science, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei, 10617, Taiwan.
Pan
Tzu-Ming
TM
D017123:Bacteriophage T7 / Q000235:genetics; D003470:Culture Media / Q000737:chemistry; D016207:Cytokines / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D051548:Histone Acetyltransferases / Q000037:antagonists & inhibitors; D001467:Hordeum / Q000737:chemistry; D007155:Immunologic Factors / Q000378:metabolism*; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013025:Glycine max / Q000737:chemistry; D014908:Triticum / Q000737:chemistry; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics
D003470:Culture Media; D016207:Cytokines; D007155:Immunologic Factors; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D051548:Histone Acetyltransferases; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases