Expression and purification of recombinant TAT-BoNT/A(1–448) under denaturing and native conditions
Expression and purification of recombinant TAT-BoNT/A(1–448) under denaturing and native conditions
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重组 TAT-BoNT/A(1–448) 在变性和天然条件下的表达和纯化
DOI：
10.1080/21655979.2016.1201252
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发表时间：
2016
期刊：
影响因子：
4.9
通讯作者：
A. I. Imani Fooladi
中科院分区：
文献类型：
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作者：
P. Saffarian;S. N. Peerayeh;J. Amani;F. Ebrahimi;Hamid Sedighianrad;R. Halabian;A. I. Imani Fooladi
A型肉毒毒素能暂时抑制肌肉收缩。目前，医生将这种毒素作为生物药物用于治疗一些肌肉收缩疾病。TAT-BoNT/A（1-448）是一种来源于肉毒杆菌毒素轻链的功能性重组蛋白。与全长肉毒杆菌毒素不同，TAT-BoNT/A（1-448）是一种自渗透分子，可以穿过生物表面，因此可以用作局部治疗剂而无需注射。为了维持TAT-BoNT/A（1-448）的功能性，有必要在表达和纯化后恢复其正常折叠。在这项研究中，我们已经调查和优化这种新的重组蛋白的表达条件。在变性条件下（ImM IPTG，在37°C），嵌合蛋白作为包涵体产生，并且需要使用变性剂（例如尿素）纯化。然而，较低的孵育温度（18°C）和较低的IPTG浓度（0.5mM）在天然条件下诱导蛋白质。在这种条件下，约60%的嵌合蛋白以可溶形式表达。
ABSTRACT Botulinum toxin type A can temporarily inhibit muscle contraction. Currently, physicians administer this toxin as a bio-drug in treatment of some muscle contraction disorders. TAT-BoNT/A(1–448) is a functional recombinant protein derived from botulinum toxin light chain. Unlike the full length botulinum toxin, TAT-BoNT/A(1–448) is a self-permeable molecule which can pass through bio-surfaces so can be used as a topical therapeutic agent without injection. To maintain the functionality of TAT-BoNT/A(1–448), it is necessary to restore its normal folding upon expression and purification. In this study, we have investigated and optimized expression conditions for this novel recombinant protein. Under denaturing condition (1 mM IPTG, at 37°C), the chimeric protein was produced as inclusion body and required to be purified using denaturing agents (e.g. urea). Yet, lower incubation temperature (18°C) and less IPTG concentration (0.5 mM) induce a protein under native condition. In such condition, about 60% of the chimeric protein was expressed in soluble form.
肉毒杆菌神经毒素是锌蛋白。
DOI：
--
发表时间：
1992
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Schiavo,G;Rossetto,O;Santucci,A;DasGupta,BR;Montecucco,C
通讯作者：
Montecucco,C