Transport proteins PotD and Crr of Escherichia coli, novel fusion partners for heterologous protein expression
Transport proteins PotD and Crr of Escherichia coli, novel fusion partners for heterologous protein expression
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DOI：
10.1016/j.bbapap.2007.09.012
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发表时间：
2007-12-01
影响因子：
3.2
通讯作者：
Lee, Jeewon
中科院分区：
文献类型：
--
作者：
Han, Kyung-Yeon;Seo, Hyuk-Seong;Lee, Jeewon
用双向凝胶电泳法(2-DE)分析了大肠杆菌蛋白质组对应激源GdnHCl的响应。我们鉴定了POTD(精胺/腐胺结合周质蛋白)和CRR(葡萄糖特异性磷酸转移酶(PTS)酶IIA组分)为应激反应蛋白。即使在胁迫条件下，可溶性蛋白质总量减少了约10%，PotD和CRR的合成也分别增加了3.5倍和2.2倍。作为融合伙伴，POTL)和CRR显著增加了许多易于聚集的异源蛋白[如人微型胰岛素原(MP-INS)、人表皮生长因子(EGF)、人促生长素原(PpGRN)、人白细胞介素2(hIL-2)、人激活诱导胞苷脱氨酶(AID)、人谷氨酸脱羧酶(GAD(448-585)、恶臭假单胞菌角质酶(CUT)、人铁蛋白轻链(hFTN-L)、人粒细胞集落刺激因子(G-CSF)和冷自体炎症综合征I蛋白(NALP3)Nacht])在大肠杆菌细胞质中的溶解度。据推测，PotD和CRR非常有效地屏蔽了与非特异性蛋白质-蛋白质相互作用相关的异源蛋白质的相互作用表面，导致包涵体的形成最有可能是由于固有的高折叠效率、伴侣样活性或两者的组合。这两种胁迫诱导蛋白都非常适合生产具有生物活性的恶臭角质酶融合突变体，有望具有生物技术和商业价值。(C)2007 Elsevier B.V.保留所有权利。
The Escherichia coli proteome response to the stressor GdnHCl was analyzed through 2-dimensional gel electrophoresis (2-DE). We identified PotD, (spermidine/putrescine-binding periplasmic protein) and Crr [glucose-specific phosphotransferase (PTS) enzyme IIA component] as a stress-responsive protein. Even under a stress situation where the total number of soluble proteins decreased by about 10%, 3.5- and 2.2-fold increase was observed in the synthesis of PotD and Crr, respectively. As fusion partners, Potl) and Crr dramatically increased the solubility of many aggregation-prone heterologous proteins [e.g. human minipro-insulin (mp-INS), human epidermal growth factor (EGF), human prepro-ghrelin (ppGRN), human interleukin-2(hIL-2), human activation induced cytidine deaminase (AID), human glutamate decarboxylase (GAD(448-585)), Pseudomonas putida cutinase (CUT), human ferritin light chain (hFTN-L), human granulocyte colony-stimulating factor (G-CSF), and cold autoinflammatory syndrome I protein (NALP3) Nacht domain (NACHT)] in the E. coli cytoplasm. Presumably PotD, and Crr were very effective in shielding interactive surfaces of heterologous proteins associated with non-specific protein-protein interactions leading to the formation of inclusion bodies most likely due to intrinsic high folding efficiency, chaperone-like activity, or a combination of both factors. Both the stress-induced proteins were well suited for the production of a biologically active fusion mutant of R putida cutinase that can be expected to be of biotechnological and commercial interest. (c) 2007 Elsevier B.V. All rights reserved.