Time- and temperature-dependent acetylation of the chemokine RANTES produced in recombinant Escherichia coli
Time- and temperature-dependent acetylation of the chemokine RANTES produced in recombinant Escherichia coli
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DOI：
10.1016/j.pep.2007.04.022
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发表时间：
2007-09-01
影响因子：
1.6
通讯作者：
Beguin, Pierre
中科院分区：
文献类型：
--
作者：
d'Alayer, Jacques;Expert-Bezancon, Nicole;Beguin, Pierre
在重组大肠杆菌BL 21（DE 3）（pDIA 17）（CCL 5-S24 F-pET-26 b）中，发现趋化因子RANTES的S24 F突变体被部分乙酰化。质谱分析和Edman测序表明，Lys-26，Lys-34，Lys-46和Lys-57对乙酰化敏感。RANTES S24 F多肽的乙酰化程度随着温度和加入诱导剂异丙基-β-D-硫代半乳糖苷（IPTG）后培养物孵育的时间而增加。这些发现表明，当假乙酰化对于产生真正的重组多肽是一个问题时，在低温和短时间段内的诱导应该是优选的。多肽的乙酰化不受分别编码乙酰辅酶A合成酶、乙酰辅酶A合成酶乙酰化酶和亚精胺乙酰转移酶的acs、yfiQ或speG缺失的影响，也不受含有编码氯霉素乙酰转移酶的cat基因的pDIA 17质粒的存在或不存在的影响。相比之下，RANTES的自发乙酰化可以通过在乙酰辅酶A存在下孵育纯化的多肽或来自诱导培养物的包涵体来证明。(C)2007年爱思唯尔公司All rights reserved.
The S24F mutant of the chemokine RANTES was found to be partly acetylated when produced in recombinant Escherichia coli BL21(DE3)(pDIA17)(CCL5-S24F-pET-26b). Mass spectrometry and Edman sequencing of peptides generated by lys-C endopeptidase indicated that Lys-26, Lys-34, Lys-46, and Lys-57 were susceptible to acetylation. The extent of acetylation of the RANTES S24F polypeptide increased with temperature and with the time during which the culture was incubated after adding the inducer isopropyl-beta-D-thiogalactoside (IPTG). These findings suggest that induction at low temperature and for a short period of time should be preferred when spurious acetylation is a problem for the production of genuine recombinant polypeptides. Acetylation of the polypeptide was not affected by deleting acs, yfiQ, or speG, which encode acetyl-CoA synthetase, acetyl-CoA synthetase acetylase, and spermidine acetyl transferase, respectively, nor by the presence or absence of the pDIA 17 plasmid, which harbours the cat gene encoding chloramphenicol acetyl transferase. By contrast, spontaneous acetylation of RANTES could be demonstrated by incubating either the purified polypeptide or inclusion bodies derived from an induced culture in the presence of acetyl-CoA. (C) 2007 Elsevier Inc. All rights reserved.