Heterologous expression of an α‑amylase inhibitor from common bean (Phaseolus vulgaris) in Kluyveromyces lactis and Saccharomyces cerevisiae
MetadatosMostrar el registro completo del ítem
Background: Phaseolamin or alpha-amylase inhibitor 1 (alpha AI) is a glycoprotein from common beans (Phaseolus vulgaris L.) that inhibits some insect and mammalian alpha-amylases. Several clinical studies support the beneficial use of bean alpha AI for control of diabetes and obesity. Commercial extracts of P. vulgaris are available but their efficacy is still under question, mainly because some of these extracts contain antinutritional impurities naturally present in bean seeds and also exhibit a lower specific activity alpha AI. The production of recombinant alpha AI allows to overcome these disadvantages and provides a platform for the large-scale production of pure and functional alpha AI protein for biotechnological and pharmaceutical applications. Results: A synthetic gene encoding alpha AI from the common bean (Phaseolus vulgaris cv. Pinto) was codon-optimised for expression in yeasts (alpha AI-OPT) and cloned into the protein expression vectors pKLAC2 and pYES2. The yeasts Kluyveromyces lactis GG799 (and protease deficient derivatives such as YCT390) and Saccharomyces cerevisiae YPH499 were transformed with the optimised genes and transformants were screened for expression by antibody dot blot. Recombinant colonies of K. lactis YCT390 that expressed and secreted functional alpha AI into the culture supernatants were selected for further analyses. Recombinant alpha AI from K. lactis YCT390 was purified using anion-exchange and affinity resins leading to the recovery of a functional inhibitor. The identity of the purified alpha AI was confirmed by mass spectrometry. Recombinant clones of S. cerevisiae YPH499 expressed functional alpha AI intracellularly, but did not secrete the protein. Conclusions: This is the first report describing the heterologous expression of the alpha-amylase inhibitor 1 (alpha AI) from P. vulgaris in yeasts. We demonstrated that recombinant strains of K. lactis and S. cerevisiae expressed and processed the alpha AI precursor into mature and active protein and also showed that K. lactis secretes functional alpha AI.
Artículo de publicación ISI
Cita del ítemMicrob Cell Fact (2017) 16:110
Los siguientes archivos de licencia se asocian a este ítem: