Research Institute for Fundamental Sciences (RIFS), University of Tabriz, Iran.
Protein J. 2011 Jun;30(5):351-8. doi: 10.1007/s10930-011-9338-9.
As a usual response, plants induce/activate various proteins which are thought to be involved in defense mechanisms against the biotic and abiotic stresses they may be confronted with. The novel DUF538 domain containing proteins with unknown functions have been found to be induced/activated in response to different environmental stress stimuli in plants. In order to perform biochemical studies with these new plant stress-responsive proteins, a cDNA containing DUF538 domain was amplified from Celosia cristata full-length leaf expression library using a specific primer set. The isolated cDNA was subsequently expressed in Escherichia coli as a part of maltose-binding fusion protein (MBP-DUF538 construct) and purified at the yield of about 32 mg per liter of cell culture by affinity chromatography without affecting the recombinant bacterial cell growth. The purified fusion product was exogenously applied (10 μg per 4 cm(2)) on the leaves of Nicotiana tobaccum L. The results revealed that fused DUF538 protein does not induce morphological reposes, but elevates redox enzyme activities including catalase, peroxidase, polyphenol oxidase and phenyalanine ammonia lyase. This is the first time ever time report with respect to the heterologous expression of a plant stress-responsive DUF538 domain that may provide a basis to study its physiological roles and biochemical activities in vitro and in vivo.
作为一种常见的反应，植物会诱导/激活各种被认为参与防御机制的蛋白质，以应对可能面临的生物和非生物胁迫。具有未知功能的新型 DUF538 结构域包含蛋白已被发现可响应植物不同的环境胁迫刺激而被诱导/激活。为了对这些新的植物应激响应蛋白进行生化研究，我们使用特定的引物从鸡冠花全长叶表达文库中扩增出含有 DUF538 结构域的 cDNA。随后，该分离的 cDNA 在大肠杆菌中表达为麦芽糖结合融合蛋白（MBP-DUF538 构建体）的一部分，并通过亲和层析以约 32 mg/L 细胞培养物的产量进行纯化，而不影响重组细菌细胞的生长。纯化的融合产物（10 μg/4 cm2）被外源性施加到烟草叶片上。结果表明，融合的 DUF538 蛋白不会诱导形态反应，但会提高氧化还原酶活性，包括过氧化氢酶、过氧化物酶、多酚氧化酶和苯丙氨酸解氨酶。这是首次报道植物应激响应 DUF538 结构域的异源表达，这可能为研究其生理功能和体外及体内的生化活性提供基础。
