Secretory Expression of Clostridium histolyticum Collagenase H in Escherichia coli
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摘要: 溶组织梭菌Clostridium histolyticum胶原酶H（ColH）作用于胶原蛋白的Y-Gly处，将其水解成小分子肽。通过在colH基因中融合大肠杆菌外膜蛋白A的信号肽序列，成功构建了分泌高分子量的ColH（116 kDa）的菌株，结果发现信号肽在N端的位置影响其分泌效果，N端若有多余的氨基酸片段会显著降低信号肽引导胶原酶的分泌功能。通过正交实验与单因素实验对诱导条件和培养基添加剂优化提高其分泌表达量, 结果为：在诱导温度为25 ℃、诱导菌浓OD600=0.9、IPTG浓度为0.1 mmol/L、装液量为20%，镁离子浓度为10 mmol/L，诱导2.5 h后添加2%甘氨酸条件下，诱导培养20 h，胞外胶原酶活性最高为0.68 U/mL，是优化前的38.1倍，显著提高了分泌表达量。研究结果还发现，镁离子的添加可增加ColH的胞外分泌。Abstract: Clostridium histolyticum collagenase H (ColH) recognizes the Y-Gly of collagen and hydrolyzes it into small peptides. The high molecular weight ColH(116 kDa) secreting strain was successfully constructed by fusing colH gene with signal peptide sequence of outer membrane protein A. In this study, we found that the secretion of ColH was effected by the position of the signal peptide at the N-terminal, and the presence of excess amino acid fragments at the N-terminal significantly reduced the secretion function of the signal peptide-guided collagenase. Orthogonal experiment and single factor experiment were used to optimize the induction conditions and medium additives to improve the secretory expression. Under the conditions of inducing temperature of 25 ℃, the cell density(OD600) of 0.9, IPTG concentration of 0.1 mmol/L, liquid volume of 20%, magnesium ion concentration of 10 mmol/L, and 2% glycine added at 2.5 h after induction, the highest extracellular collagenase activity was 0.68 U/mL after induction for 20 h, which was 38.1 times of that before optimization, and the secretory expression level was greatly increased. Glycine added into the culture medium is a common strategy to promote the secretion of recombinant protein. Experimental results showed that the amount and time of glycine added after induction had the greatest influence on the secretion of collagenase. The addition of calcium and magnesium ions in the medium can promote the growth of E.coli, the results also showed that but only the addition of magnesium ion can promote the secretion of ColH.
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图 1 构建的两种ColH分泌质粒结构示意图
Figure 1. Schematic representation of two ColH secretory expression plasmids.
图 2 三种重组菌株胶原酶的胞外分泌情况分析
Figure 2. Extracellular secretion of collagenase of the three strains. A: SDS-PAGE analysis of the extracellular component at 10% gel concentration; B: enzymatic activity of the extracellular component hydrolyzing PZ peptide. 1: protobacteria, 2: strain A, 3: strain B.
图 4 SDS-PAGE电泳分析胶原酶ColH的分泌表达鉴定（A，胞外分泌蛋白分析；B，胞内蛋白分析）
Figure 4. SDS-PAGE analysis of culture medium components and intracellular soluble components
图 5 甘氨酸添加量对ColH表达和分泌的影响
Figure 5. Effects of amount of glycine added on ColH expression and secretion
图 6 甘氨酸添加时间对ColH表达和分泌的影响
Figure 6. Effect of time of glycine added on ColH expression and secretion
图 7 钙离子对ColH表达和分泌的影响
Figure 7. Effects of calcium ion on ColH expression and secretion.
图 8 镁离子对ColH表达和分泌的影响
Figure 8. Effects of magnesium ion on ColH expression and secretion.
图 9 诱导时间对ColH表达和分泌的影响
Figure 9. Effect of inducing time on ColH expression and secretion.
表 1 本研究使用的引物
Table 1. Primers used in this work. The underlined part is the restriction site, the oblique part is the signal peptide sequence, and the bold part is the homologous sequence of primer and carrier terminal.
Primer names Primer sequence（5′–3′） F1 ATCGAGGATCCATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGG
TTTCGCTACCGTAGCGCAGGCCGTCCAGAACGAAAGCAAACGCTACR1 ATCGACTCGAGACGACCAACGCTGCCTTC F2 AAGGAGATATACCATGAAAAAGACAGCTATCGCGATTGC R2 GGTGGTGGTGCTCGAGACGAC 
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表 2 因素水平表
Table 2. Factor level table
Factors Levels Inducing
temperature/℃C(IPTG)/
(mmol·L−1)OD600 Loading
volume/%1 20 0.1 0.45 10 2 25 0.5 0.90 20 3 30 1.0 1.80 30
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