Veisi Kamal, Farajnia Safar, Zarghami Nosratollah, Khoram Khorshid Hamid Reza, Samadi Nasser, Ahdi Khosroshahi Shiva, Zarei Jaliani Hossein
Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Department of Medical Biotechnologies, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran. ; Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran.
Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Adv Pharm Bull. 2015 Dec;5(Suppl 1):621-7. doi: 10.15171/apb.2015.084. Epub 2015 Dec 31.
Formation of inclusion bodies is a considerable obstacle threatening the advantages of E. coli expression system to serve as the most common and easiest system in recombinant protein production. To solve this problem, several strategies have been proposed among which application of molecular chaperones is of remarkable consideration. The aim of this study was to evaluate the effects of molecular chaperones on soluble expression of aggregation-prone humanized single chain antibody.
To increase the solubility of a humanized single chain antibody (hscFv), different chaperone plasmids including PG-tf2 (GroES- GroEL- tig), ptf16 (tig) and pGro7 (GroES- GroEL) were co-expressed in BL21 cells containing pET-22b- hscFv construct. The solubility of recombinant hscFv was analyzed by SDS-PAGE. After purification of soluble hscFv by Ni-NTA column, the biological activity and cytotoxicity of the recombinant protein were tested by ELISA and MTT assay, respectively.
SDS-PAGE analysis of the hscFv revealed that chaperone utility remarkably increased (up to 50%) the solubility of the protein. ELISA test and MTT assay analyses also confirmed the biological activity of the gained hscFv in reaction with A431 cells (OD value: 2.6) and inhibition of their proliferation, respectively.
The results of this study revealed that co-expression of chaperones with hscFv leads to remarkable increase in the solubility of the recombinant hscFv, which could be of great consideration for large scale production of recombinant single chain antibodies.
包涵体的形成是一个相当大的障碍，威胁着大肠杆菌表达系统作为重组蛋白生产中最常见、最简单系统的优势。为了解决这个问题，已经提出了几种策略，其中分子伴侣的应用受到了显著关注。本研究的目的是评估分子伴侣对易于聚集的人源化单链抗体可溶性表达的影响。
为了提高人源化单链抗体（hscFv）的溶解度，将不同的伴侣质粒，包括PG-tf2（GroES - GroEL - tig）、ptf16（tig）和pGro7（GroES - GroEL），与含有pET - 22b - hscFv构建体的BL21细胞共表达。通过SDS - PAGE分析重组hscFv的溶解度。通过镍 - 氮三乙酸（Ni - NTA）柱纯化可溶性hscFv后，分别通过酶联免疫吸附测定（ELISA）和噻唑蓝（MTT）比色法检测重组蛋白的生物活性和细胞毒性。
对hscFv的SDS - PAGE分析表明，伴侣蛋白的使用显著提高（高达50%）了该蛋白的溶解度。ELISA试验和MTT分析还分别证实了获得的hscFv与A431细胞反应的生物活性（光密度值：2.6）及其对细胞增殖的抑制作用。
本研究结果表明，伴侣蛋白与hscFv共表达可显著提高重组hscFv的溶解度，这对于重组单链抗体的大规模生产具有重要意义。
