Expression, purification and characterization of ProL protein in the endophytic fungus Cytospora rhizophorae from Morinda officinalis
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摘要:目的
从巴戟天内生真菌Cytospora rhizophorae中克隆proL基因，异源表达ProL蛋白并研究其理化性质，为解析ProL在合成Cytorhizins类新骨架活性化合物途径中的生物学功能奠定基础。
方法利用PCR技术从C. rhizophorae中克隆proL基因，通过同源重组的方法将proL基因片段插入到pET28a原核表达载体，在大肠埃希菌Escherichia coli中异源表达，使用尿素对ProL蛋白梯度复性，采用SDS-PAGE技术和质谱测序对ProL蛋白进行分析和鉴定，运用生物信息学方法对ProL蛋白的氨基酸序列相似性进行分析，推测其编码蛋白的结构和功能。
结果克隆得到proL基因的编码序列，开放阅读框全长909 bp，编码303个氨基酸，ProL蛋白分子式为C1495H2320N424O444S13，相对分子质量为33 754.22，原子数为4 696，等电点为5.69，为酸性蛋白。ProL蛋白在大肠埃希菌中以包涵体的形式大量表达，尿素梯度复性获得了纯度为98.9%的ProL蛋白。生物信息学分析发现ProL氨基酸序列在已知蛋白中与Aspergillus ibericus XP025570169.1的酰胺水解酶2相似性最高，为59.40%，其三维结构模型中包含8个α−螺旋和8个β−折叠，保守氨基酸序列为207~216位。
结论ProL蛋白属于酰胺水解酶超家族，预测其为一种新颖蛋白，该蛋白可能在生成高度氧化的二苯甲酮类化合物途径中起水解作用。
Abstract:ObjectiveTo clone the coding sequence of proL gene in the endophytic fungus Cytospora rhizophorae derived from Morinda officinalis, obtain ProL protein by heterologous expression and investigate its physicochemical properties, thereby providing a basis for the subsequent research on biological function of the ProL in the biosynthesis pathway of new bioactive compounds cytorhizins.
MethodThe proL gene from C. rhizophorae was amplified by PCR, the proL gene fragment was inserted into the prokaryotic expression vector of pET28a by the homologous recombination method and heterologously expressed in Escherichia coli. The ProL protein was renatured by refolding buffer containing urea with a gradiently decreased concentration, and SDS-PAGE analysis and mass spectrometry sequencing were used to verify the target ProL protein. The bioinformatic methods were employed to analyze the similarity of ProL protein with other related proteins, and predict the structure and function of ProL protein.
ResultThe coding sequence of proL gene was cloned, the open reading frame of proL gene is 909 bp in length, which encodes 303 amino acids, the molecular formula of ProL is C1495H2320N424O444S13, the relative molecular weight is 33 754.22, the total number of atoms is 4 696, PI is 5.69, so ProL is an acidic protein. ProL protein was abundantly expressed as inclusion bodies in E.coli, the recombinant ProL was obtained with a purity of 98.9%. Bioinformatics analysis results indicated that ProL protein had the highest amino acid sequence similarity (59.40%) with amidohydrolase 2 from Aspergillus ibericus XP025570169.1. The three-dimensional structure model of ProL protein is composed of eight α-helixes and eight β-folds. The conserved amino acid sequence is located at the position from 207 to 216.
ConclusionsProL protein belongs to the amidohydrolase superfamily, and is predicted as a novel protein. ProL protein might play a role of hydrolysis in the biosynthesis pathway of highly oxidized benzophenones.
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Keywords:
- Morinda officinalis /
- Cytospora rhizophorae /
- proL gene /
- cloning /
- expression /
- bioinformatic
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图 1 目的基因proL的PCR验证
A：proL基因扩增； B：菌液PCR验证； M：DNA marker 2k plus；1：proL基因；2~11：pET28a-proL克隆子
Figure 1. Verification of target gene proL by PCR
A: proL gene amplification; B: Bacterial liquid verification by PCR; M: DNA marker 2k plus; 1: proL gene; 2-11: Clones of pET28a-proL
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图 2 无内含子的pET28a-proL重组质粒构建
M：DNA marker 2k plus；1、2：pET28a-proL中的片段1和2；3：无内含子的目的基因
Figure 2. Construction of pET28a-proL recombinant plasmids with intron excision
M: DNA marker 2k plus; 1, 2: Fragments 1 and 2 of pET28a-proL; 3: Target gene with intron excision
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图 4 基于水解酶序列的真菌系统进化树
Figure 4. Phylogenetic tree of fungi based on their hydrolase sequences
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图 5 ProL蛋白的表达纯化
A：蛋白表达；B：蛋白纯化；C：蛋白脱盐；M: Blue plus marker；1：未诱导总蛋白；2：37 ℃诱导上清；3：37 ℃诱导沉淀；4：25 ℃诱导上清；5：25 ℃诱导沉淀；6：透析前样品；7：透析后样品；8：His-Ni柱穿过液；9：68 mmol/L咪唑洗脱液(峰1)；10：68 mmol/L咪唑洗脱液(峰2)； 11：68 mmol/L咪唑洗脱液脱盐(峰1)；12: 68 mmol/L咪唑洗脱液脱盐(峰2)
Figure 5. Expression and purification of ProL protein
A: Protein expression; B: Protein purification; C: Protein desalination; M: Blue plus marker; 1:Uninducedtotal protein; 2: Supernatant incubated at 37 ℃; 3: Precipitation incubated at 37 ℃; 4: Supernatant incubated at 25 ℃; 5: Precipitation incubated at 25 ℃; 6: Sample before dialysis; 7: Sample after dialysis; 8: His Ni-column passing through liquid; 9: 68 mmol/L imidazole eluatetion (peak one); 10: 68 mmol/L imidazole eluate (peak two); 11: Desalted sample of 68 mmol/L imidazole eluate (peak one); 12: Desalted sample of 68 mmol/L imidazole eluate (peak two)
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图 6 ProL蛋白质谱测序结果
灰色阴影部分为ProL氨基酸序列
Figure 6. Mass spectrometry sequencing results of ProL protein
Sequence with grey background is amino acid sequence of ProL
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图 8 Cytospora rhizophorae A761中二苯甲酮类化合物生物合成途径预测
1：乙酰辅酶A；2：Endocrocinanthrone前体；3：Emodin前体；4：1,2,3,8-tetrahydroxy-6-methylanthracene-9,10-dione；5：1,8,9,10-tetrahydroxy-3-methyldibenzo[b,e]oxepine-6,11-dione；6：Cytosporaphenone A；PKs：聚酮合酶；[O]表示由单加氧酶催化
Figure 8. Prediction of the biosynthesis pathway of benzophenones in Cytospora rhizophorae A761
1: Acetyl coenzyme A; 2: Precursor of endocrocinanthrone; 3: Precursor of emodin; 4: 1,2,3,8-tetrahydroxy-6-methylanthracene-9,10-dione; 5: 1,8,9,10-tetrahydroxy-3-methyldibenzo[b,e]oxepine-6,11-dione; 6: Cytosporaphenone A; PKs: Polyketide synthase; [O] indicates being catalyzed by monooxygenase
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