Prokaryotic Expression and Acetylation of AphB Protein of Vibrio alginolyticus
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摘要:
目的 研究LysR家族转录因子AphB蛋白是否存在乙酰化修饰，为进一步研究乙酰化修饰对该蛋白功能的影响提供理论基础。 方法 以溶藻弧菌（Vibrio alginolyticus）HY9901 LysR家族转录因子AphB为对象，根据GenBank上溶藻弧菌aphB序列（No.WP_005380599.1）设计引物，采用大肠杆菌表达体系异源诱导表达，设置时间、温度和IPTG浓度梯度优化表达条件，最后纯化AphB蛋白，通过抗乙酰赖氨酸特异性抗体验证AphB蛋白的乙酰化修饰程度。 结果 aphB全长约876 bp，37 ℃时菌液加入0.1 mmol·L−1的IPTG诱导6 h后，AphB蛋白的表达量最高，纯化后的蛋白大小为37.3 kD，AphB自身存在乙酰化修饰位点，但其乙酰化程度在体外不受脱乙酰酶CobB的调控。 结论 初步证明了AphB蛋白是一种乙酰化蛋白且在体外不能被脱乙酰酶CobB脱乙酰化，研究结论丰富了原核生物弧菌中乙酰化修饰的相关理论，为溶藻弧菌毒力基因aphB的翻译后调控机制研究提供了科学参考。 Abstract:Objective Prokaryotic expression and acetylation modification of the LysR family transcription factor AphB protein were studied. Method Primers of AphB of Vibrio alginolyticus HY9901 were designed based on the GenBank database No:WP_005380599.1. Heterologous induction was carried out by using E. coli expression system with optimized time, temperature, and IPTG concentration gradient. AphB protein was then purified to determine the degree of acetylation modification using anti-acetyl lysine specific antibody. Result The full length of aphB gene was approximately 876 bp. After the bacterial solution was induced with 0.1 mmol·L−1 of IPTG at 37 ℃ for 6 h, the expression of the fusion protein peaked. The purified fusion protein was 37.3 kD with an acetylation modification site. However, in vitro, the degree of acetylation was not regulated by deacetylase CobB. Conclusion This study preliminarily determined AphB protein to be an acetylated protein that could not be enzymatically deacetylated by deacetylase CobB in vitro. The results reinforced the existing theory of acetylation modification in prokaryotic Vibrio and provided a valuable reference for studying the post-translation regulation mechanism of virulence gene aphB of V. alginolyticus. -
图 1 aphB基因克隆鉴定
M：DNA Marker；1～4：目的基因扩增结果。
Figure 1. Identification of cloned aphB gene
M: DNA marker; 1-4: Amplified target gene.
图 2 pET-28a-aphB构建的菌落PCR分析
M：DNA Marker；1～4：目的基因扩增结果。
Figure 2. PCR of colony constructed by pET-28a-aphB
M: DNA marker; 1-4: Amplified target gene.
图 3 AphB蛋白SDS-PAGE 电泳分析
M：蛋白质Marker；1：pET-28a-aphB（诱导）；2：pET-28a-aphB（未诱导）；3：pET-28a（诱导）；4：pET-28a（未诱导）。
Figure 3. SDS-PAGE electrophoresis of AphB protein
M: Protein marker; 1: pET-28a-aphB (induced); 2: pET-28a-aphB (uninduced); 3: pET-28a (induced); 4: pET-28a (uninduced).
图 4 AphB融合蛋白在不同诱导温度的表达
M：蛋白质Marker；1：全菌蛋白（28 ℃）；2：可溶性蛋白（28 ℃）：3：包涵体蛋白（28 ℃）；4：全菌蛋白（37 ℃）；5：可溶性蛋白（37 ℃）；6：包涵体蛋白（37 ℃）。
Figure 4. Expressions of AphB fusion protein at different induction temperatures
M: Protein marker; 1: Whole bacterial protein (28 ℃); 2: Soluble body proteins (28 ℃); 3: Inclusion body proteins (28 ℃); 4: Whole bacterial protein (37 ℃); 5: Soluble body proteins (37 ℃); 6: Inclusion body proteins (37 ℃).
图 5 AphB全菌蛋白在不同诱导时间的表达
M：蛋白质Marker；1～8：诱导时间为 1、2、3、4、5、6、7 、8 h。
Figure 5. Expressions of AphB whole bacterial protein at different induction times
M: Protein marker; 1–8: Induction times of 1, 2, 3, 4, 5, 6, 7, and 8 h, respectively.
图 6 AphB全菌蛋白在不同IPTG诱导浓度的表达
M：蛋白质Marker；1~8：IPTG 浓度为0、0.1、0.2、0.4、0.6、0.8、1.0、2.0 mmol·L−1。
Figure 6. Expressions of AphB whole bacterial protein at different IPTG induced concentrations
M: Protein marker; 1–8: IPTG concentrations of 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 2.0 mmol·L−1, respectively.
图 7 AphB蛋白的纯化
M：蛋白质Marker；1～2：第1、2次蛋白流穿液；3～4：第1、2次蛋白洗涤液；5～9：第1～5次蛋白洗脱液。
Figure 7. Purification of AphB protein
M: Protein marker; 1–2：Protein flow-through buffer of 1st and 2nd times; 3–4: Protein washing buffer of 1st and 2nd times; 5–9: Protein elution buffer of 1–5 times.
图 8 Western Blot鉴定融合蛋白AphB的乙酰化
M：蛋白质Marker；1：无脱乙酰酶CobB、无NAD+组；2：有脱乙酰酶CobB、无NAD+组；3：无脱乙酰酶CobB、有NAD+组；4：有脱乙酰酶CobB、有NAD+组。
Figure 8. Identification of acetylation of recombinant protein AphB by western blot
M: Protein marker; 1: No deacetylase CobB, no NAD+ group; 2: Deacetylase CobB, no NAD+ group; 3: No deacetylase CobB, NAD+ group; 4: Deacetylase CobB, NAD+ group.
表 1 引物序列
Table 1. Primer sequences
引物
Primer核酸序列 (5′-3′)
Nucleotide sequence (5′-3′)aphB-F CCGGAATTCATGAAATTAGATGACTTAAACCTGT aphB-T CCGCTCGAGTTAGTGAATGTTATAAGCGATCACA T7 TGCTAGTTATTGCTCAGCGG T7-Term TAATACGACTCACTATAGGG 下划线表示酶切位点，aphB-F：EcoR I，aphB-T: Xho I。
Underline indicates the restriction site, aphB-F：EcoR I，aphB-T: Xho I.
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