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Soluble Expression and Catalytic Properties of Codon-Optimized Recombinant Bromelain from MD2 Pineapple in Escherichia coli
The Protein Journal ( IF 3 ) Pub Date : 2021-03-13 , DOI: 10.1007/s10930-021-09974-9
Rafida Razali 1 , Cahyo Budiman 1 , Khairul Azfar Kamaruzaman 1 , Vijay Kumar Subbiah 1
Affiliation  

Bromelain, a member of cysteine proteases, is found abundantly in pineapple (Ananas comosus), and it has a myriad of versatile applications. However, attempts to produce recombinant bromelain for commercialization purposes are challenging due to its expressibility and solubility. This study aims to express recombinant fruit bromelain from MD2 pineapple (MD2Bro; accession no: OAY85858.1) in soluble and active forms using Escherichia coli host cell. The gene encoding MD2Bro was codon-optimized, synthesized, and subsequently ligated into pET-32b( +) for further transformation into Escherichia coli BL21-CodonPlus(DE3). Under this strategy, the expressed MD2Bro was in a fusion form with thioredoxin (Trx) tag at its N-terminal (Trx-MD2Bro). The result showed that Trx-MD2Bro was successfully expressed in fully soluble form. The protein was successfully purified using single-step Ni2+-NTA chromatography and confirmed to be in proper folds based on the circular dichroism spectroscopy analysis. The purified Trx-MD2Bro was confirmed to be catalytically active against N-carbobenzoxyglycine p-nitrophenyl ester (N-CBZ-Gly-pNP) with a specific activity of 6.13 ± 0.01 U mg−1 and inhibited by a cysteine protease inhibitor, E-64 (IC50 of 74.38 ± 1.65 nM). Furthermore, the catalytic efficiency (kcat/KM) Trx-MD2Bro was calculated to be at 5.64 ± 0.02 × 10–2 µM−1 s−1 while the optimum temperature and pH were at 50 °C and pH 6.0, respectively. Furthermore, the catalytic activity of Trx-MD2Bro was also affected by ethylenediaminetetraacetic acid (EDTA) or metal ions. Altogether it is proposed that the combination of codon optimization and the use of an appropriate vector are important in the production of a soluble and actively stable recombinant bromelain.



中文翻译:

MD2菠萝密码子优化重组菠萝蛋白酶在大肠杆菌中的可溶性表达及催化特性

菠萝蛋白酶是半胱氨酸蛋白酶的一员,在菠萝 ( Ananas comosus ) 中含量丰富,具有多种用途。然而,由于其可表达性和溶解性,生产用于商业化目的的重组菠萝蛋白酶的尝试具有挑战性。本研究旨在使用大肠杆菌 宿主细胞以可溶性和活性形式表达来自 MD2 菠萝(MD2Bro;登录号:OAY85858.1)的重组水果菠萝蛋白酶 。编码 MD2Bro 的基因经过密码子优化、合成,随后连接到 pET-32b(+) 以进一步转化 大肠杆菌 BL21-CodonPlus(DE3)。在这种策略下,表达的 MD2Bro 呈融合形式,在其 N 端 (Trx-MD2Bro) 带有硫氧还蛋白 (Trx) 标签。结果表明Trx-MD2Bro以完全可溶的形式成功表达。使用单步 Ni 2+ -NTA 色谱法成功纯化了蛋白质,并根据圆二色光谱分析确认蛋白质处于适当的折叠状态。经证实,纯化的 Trx-MD2Bro 对 N-羧基苯甲氧基甘氨酸硝基苯酯 (N-CBZ-Gly-pNP) 具有催化活性,比活性为 6.13 ± 0.01 U mg -1并被半胱氨酸蛋白酶抑制剂 E-抑制64 (IC 50为 74.38 ± 1.65 nM)。此外,催化效率(ķ CA/K M ) Trx-MD2Bro 计算为 5.64 ± 0.02 × 10 –2  µM -1  s -1而最佳温度和 pH 值分别为 50 °C 和 pH 6.0。此外,Trx-MD2Bro 的催化活性也受到乙二胺四乙酸 (EDTA) 或金属离子的影响。总而言之,密码子优化和适当载体的使用的组合在可溶性和活性稳定的重组菠萝蛋白酶的生产中很重要。

更新日期:2021-03-15
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