Jensen Johanne Mørch, Vester-Christensen Malene Bech, Møller Marie Sofie, Bønsager Birgit C, Christensen Hans Erik Mølager, Hachem Maher Abou, Svensson Birte
Enzyme and Protein Chemistry, Department of Systems Biology, Søltofts Plads Building 224, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark.
Protein Expr Purif. 2011 Oct;79(2):217-22. doi: 10.1016/j.pep.2011.04.009. Epub 2011 Apr 27.
The limit dextrinase inhibitor (LDI) from barley seeds acts specifically on limit dextrinase (LD), an endogenous starch debranching enzyme. LDI is a 14 kDa hydrophobic protein containing four disulfide bonds and one unpaired thiol group previously found to be either glutathionylated or cysteinylated. It is a member of the so-called CM-protein family that includes α-amylase and serine protease inhibitors, which have been extremely challenging to produce recombinantly in functional form and in good yields. Here, LDI is produced in very high yields by secretory expression by Pichia pastoris applying high cell-density fermentation in a 5L fed-batch bioreactor. Thus about 200mg of LDI, which showed twofold higher inhibitory activity towards LD than LDI from barley seeds, was purified from 1L of culture supernatant by His-tag affinity chromatography and gel filtration. Electrospray ionization mass spectrometry verified the identity of the produced glutathionylated LDI-His(6). At a 1:1M ratio the recombinant LDI completely inhibited hydrolysis of pullulan catalyzed by 5-10 nM LD. LDI retained stability in the pH 2-12 range and at pH 6.5 displayed a half-life of 53 and 33 min at 90 and 93°C, respectively. The efficient heterologous production of LDI suggests secretory expression by P. pastoris to be a promising strategy to obtain other recombinant CM-proteins.
大麦种子中的极限糊精酶抑制剂(LDI)特异性作用于极限糊精酶(LD),一种内源性淀粉脱支酶。LDI是一种14 kDa的疏水蛋白,含有四个二硫键和一个未配对的硫醇基团,此前发现其要么被谷胱甘肽化,要么被半胱氨酸化。它是所谓CM蛋白家族的成员,该家族包括α-淀粉酶和丝氨酸蛋白酶抑制剂,以功能形式和高产量进行重组生产极具挑战性。在这里,通过在5L补料分批生物反应器中应用高细胞密度发酵,毕赤酵母分泌表达以非常高的产量生产LDI。因此,从1L培养上清液中通过His标签亲和色谱和凝胶过滤纯化得到约200mg的LDI,其对LD的抑制活性比大麦种子中的LDI高两倍。电喷雾电离质谱验证了所产生的谷胱甘肽化LDI-His(6)的身份。以1:1M的比例,重组LDI完全抑制了5-10 nM LD催化的支链淀粉水解。LDI在pH 2-12范围内保持稳定,在pH 6.5时,在90°C和93°C下的半衰期分别为53分钟和33分钟。LDI的高效异源生产表明毕赤酵母分泌表达是获得其他重组CM蛋白的一种有前景的策略。