凝集素是医学和药理学应用中的重要蛋白质。来自天然来源的凝集素中的杂质和通过重组技术产生非活性蛋白是使用凝集素的主要障碍。表达具有串联重复结构的重组凝集素可以潜在地克服这些问题，但是很少有研究系统地研究了这种可能性。在本研究中，使用三种不同形式的来自Bryopsisplumosa（BPL2）的重组甘露糖结合凝集素进行了研究-即，单体（rD1BPL2），以及二聚体（rD2BPL2），和四聚体(rD4BPL2)排列为串联重复。诱导分子异丙基β-D-1-硫代吡喃半乳糖苷的浓度和诱导时间对每种构建体的表达效率没有影响。在测试的结构中，只有rD4BPL2对马红细胞具有血凝活性；对前者的活性比天然BPL2高64倍。重组和天然BPL2显示出碳水化合物特异性的差异;rD4BPL2的活性被糖蛋白胎球蛋白抑制,而天然BPL2也被d-甘露糖抑制。我们的结果表明，作为串联重复序列的表达可以使用细菌表达系统大规模提高凝集素生产的效率。
Lectin is an important protein in medical and pharmacological applications. Impurities in lectin derived from natural sources and the generation of inactive proteins by
recombinant technology are major obstacles for the use of lectins. Expressing
recombinant lectin with a tandem repeat structure can potentially overcome these problems, but few studies have systematically examined this possibility. This was investigated in the present study using three distinct forms of
recombinant mannose-binding lectin from Bryopsis plumosa (BPL2)-i.e., the monomer (rD1BPL2), as well as the dimer (rD2BPL2), and tetramer (rD4BPL2) arranged as tandem repeats. The concentration of the inducer molecule isopropyl β-D-1-thiogalactopyranoside and the induction time had no effect on the efficiency of the expression of each construct. Of the tested constructs, only rD4BPL2 showed hemagglutination activity towards horse erythrocytes; the activity of towards the former was 64 times higher than that of native BPL2.
Recombinant and native BPL2 showed differences in carbohydrate specificity; the activity of rD4BPL2 was inhibited by the glycoprotein fetuin, whereas that of native BPL2 was also inhibited by d-mannose. Our results indicate that expression as tandem repeat sequences can increase the efficiency of lectin production on a large scale using a bacterial expression system.
PDF(Sci-hub)