Cardoso Vânia, Brás Joana L A, Costa Inês F, Ferreira Luís M A, Gama Luís T, Vincentelli Renaud, Henrissat Bernard, Fontes Carlos M G A
Centro de Investigação Interdisciplinar em Sanidade Animal-Faculdade de Medicina Veterinária, Universidade de Lisboa, Pólo Universitário do Alto da Ajuda, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal.
NZYTech Ltd., Estrada do Paço do Lumiar, Campus do Lumiar, 1649-038 Lisboa, Portugal.
Int J Mol Sci. 2022 Apr 5;23(7):4024. doi: 10.3390/ijms23074024.
In nature, the deconstruction of plant carbohydrates is carried out by carbohydrate-active enzymes (CAZymes). A high-throughput (HTP) strategy was used to isolate and clone 1476 genes obtained from a diverse library of recombinant CAZymes covering a variety of sequence-based families, enzyme classes, and source organisms. All genes were successfully isolated by either PCR (61%) or gene synthesis (GS) (39%) and were subsequently cloned into Escherichia coli expression vectors. Most proteins (79%) were obtained at a good yield during recombinant expression. A significantly lower number (p < 0.01) of proteins from eukaryotic (57.7%) and archaeal (53.3%) origin were soluble compared to bacteria (79.7%). Genes obtained by GS gave a significantly lower number (p = 0.04) of soluble proteins while the green fluorescent protein tag improved protein solubility (p = 0.05). Finally, a relationship between the amino acid composition and protein solubility was observed. Thus, a lower percentage of non-polar and higher percentage of negatively charged amino acids in a protein may be a good predictor for higher protein solubility in E. coli. The HTP approach presented here is a powerful tool for producing recombinant CAZymes that can be used for future studies of plant cell wall degradation. Successful production and expression of soluble recombinant proteins at a high rate opens new possibilities for the high-throughput production of targets from limitless sources.
在自然界中，植物碳水化合物的解构是由碳水化合物活性酶（CAZymes）完成的。采用高通量（HTP）策略从涵盖各种基于序列的家族、酶类和来源生物的重组CAZymes多样文库中分离并克隆了1476个基因。所有基因均通过PCR（61%）或基因合成（GS）（39%）成功分离，随后克隆到大肠杆菌表达载体中。大多数蛋白质（79%）在重组表达过程中获得了较高产量。与细菌来源的蛋白质（79.7%）相比，真核生物（57.7%）和古细菌（53.3%）来源的蛋白质可溶性显著降低（p < 0.01）。通过GS获得的基因产生的可溶性蛋白质数量显著减少（p = 0.04），而绿色荧光蛋白标签提高了蛋白质的可溶性（p = 0.05）。最后，观察到氨基酸组成与蛋白质可溶性之间的关系。因此，蛋白质中非极性氨基酸比例较低且带负电荷氨基酸比例较高可能是其在大肠杆菌中具有较高可溶性的良好预测指标。本文介绍的HTP方法是生产重组CAZymes的有力工具，可用于未来植物细胞壁降解研究。以高比率成功生产和表达可溶性重组蛋白为从无限来源高通量生产目标开辟了新的可能性。
