Brain-Isasi Stephanie, Álvarez-Lueje Alejandro, Higgins Thomas Joseph V
Drug Analysis Laboratory, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, Chile.
CSIRO Agriculture and Food, GPO Box 1700, Canberra, ACT, 2601, Australia.
Microb Cell Fact. 2017 Jun 15;16(1):110. doi: 10.1186/s12934-017-0719-4.
Phaseolamin or α-amylase inhibitor 1 (αAI) is a glycoprotein from common beans (Phaseolus vulgaris L.) that inhibits some insect and mammalian α-amylases. Several clinical studies support the beneficial use of bean αAI 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 αAI. The production of recombinant αAI allows to overcome these disadvantages and provides a platform for the large-scale production of pure and functional αAI protein for biotechnological and pharmaceutical applications.
A synthetic gene encoding αAI from the common bean (Phaseolus vulgaris cv. Pinto) was codon-optimised for expression in yeasts (αAI-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 αAI into the culture supernatants were selected for further analyses. Recombinant αAI 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 αAI was confirmed by mass spectrometry. Recombinant clones of S. cerevisiae YPH499 expressed functional αAI intracellularly, but did not secrete the protein.
This is the first report describing the heterologous expression of the α-amylase inhibitor 1 (αAI) from P. vulgaris in yeasts. We demonstrated that recombinant strains of K. lactis and S. cerevisiae expressed and processed the αAI precursor into mature and active protein and also showed that K. lactis secretes functional αAI.
菜豆素或α-淀粉酶抑制剂1（αAI）是一种来自普通菜豆（Phaseolus vulgaris L.）的糖蛋白，可抑制某些昆虫和哺乳动物的α-淀粉酶。多项临床研究支持使用菜豆αAI来控制糖尿病和肥胖症。普通菜豆的商业提取物已有供应，但其功效仍受到质疑，主要是因为其中一些提取物含有菜豆种子中天然存在的抗营养杂质，并且αAI的比活性较低。重组αAI的生产可以克服这些缺点，并为大规模生产用于生物技术和制药应用的纯功能性αAI蛋白提供一个平台。
一个编码来自普通菜豆（Phaseolus vulgaris cv. Pinto）的αAI的合成基因经过密码子优化以在酵母中表达（αAI-OPT），并克隆到蛋白质表达载体pKLAC2和pYES2中。用优化后的基因转化乳酸克鲁维酵母GG799（以及蛋白酶缺陷衍生物如YCT390）和酿酒酵母YPH499，通过抗体斑点印迹筛选转化子的表达情况。选择在培养上清液中表达并分泌功能性αAI的乳酸克鲁维酵母YCT390的重组菌落进行进一步分析。使用阴离子交换树脂和亲和树脂纯化来自乳酸克鲁维酵母YCT390的重组αAI，从而获得功能性抑制剂。通过质谱确认纯化的αAI的身份。酿酒酵母YPH499的重组克隆在细胞内表达功能性αAI，但不分泌该蛋白。
这是第一份描述普通菜豆的α-淀粉酶抑制剂1（αAI）在酵母中的异源表达的报告。我们证明，乳酸克鲁维酵母和酿酒酵母的重组菌株将αAI前体表达并加工成成熟的活性蛋白，并且还表明乳酸克鲁维酵母分泌功能性αAI。
