Recombinant Expression, Refolding, and Purification of Lysobacter enzymogenes Lys-C in Escherichia coli
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摘要: 在产酶溶杆菌来源的Lys-C成熟肽序列的N端和C端分别融合人工前肽 (MGSK) 和6×His标签，将密码子优化后的序列插入表达载体pET-28a。以IPTG诱导型启动子PT7控制Lys-C的高效表达，并针对重组菌株JM109DE3_PT7-LysC开展生物反应器高密度发酵生产Lys-C。然后收集和溶解包涵体得到Lys-C变性液，通过Sephadex G25层析脱除DTT，并在Lys-C复性液中添加前导肽 (pre-N-pro) 辅助成熟肽蛋白折叠。进一步，通过切向流过滤、Ni NTA-Sepharose亲和层析和Sephacryl S-100层析等一系列纯化步骤，获得高纯度的重组Lys-C。最后进行酶切三肽底物和门冬胰岛素前体检测，分析重组Lys-C的活性水平。重组Lys-C发酵产量为2.4 g/L，经复性和纯化后的终产量可达48 mg/L。添加80 mg/L pre-N-pro促进了重组Lys-C的复性，复性后酶活相比于未添加pre-N-pro时提升了4.8倍，最高可达到13.8 AU/L。经过多步纯化后，重组Lys-C的比酶活为10.2 AU/mg，且对于门冬胰岛素前体的酶切转化率可达93.5%。Abstract: To develop a high-efficiency recombinant expression and purification strategy for lysyl endopeptidase (Lys-C), and present a new choice for breaking the bottleneck of Lys-C generated by natural strains, which has a poor yield and high cost. The artificial pro-peptide (MGSK) and the 6×His tag were fused to the N-terminus and C-terminus of the Lys-C mature peptide gene sequence from Lysobacter enzymogenes, respectively, and then the codon-optimized sequence was inserted into plasmid pET-28a. The Lys-C was efficiently expressed in the recombinant strain JM109DE3_PT7- LysC and controlled by the IPTG-inducible promoter PT7. The inclusion bodies were collected by high-density fermentation in a bioreactor, after which they were solubilized to obtain the Lys-C denaturing solution and DTT was removed by Sephadex G25 chromatography. Then the pre-pro peptide (pre-N-pro) was added to Lys-C refolding solution to assist in the folding of the mature protein. Further, the large volume Lys-C refolding solution was concentrated using tangential flow filtration, followed by a multi-step purification process of Ni NTA-Sepharose affinity chromatography, ultrafiltration, and Sephacryl S-100 chromatography to obtain high-purity recombinant Lys-C. Finally, the activity of recombinant Lys-C was measured by chromogenic reaction and digestion of insulin aspart precursors. The recombinant Lys-C fermentation yield was achieved at 2.4 g/L, and the final yield reached 48 mg/L after renaturation and purification. The Lys-C enzyme activity was improved by addition of 80 mg/L of pre-N-pro and reached up to 13.8 AU/L, which increased by 4.8-fold. The specific enzyme activity of Lys-C achieved 10.2 AU/mg after multi-step purification. And the digestion efficiency of Lys-C to insulin precursors reached 93.5%. In this study, recombinant Lys-C with high yield and good activity was obtained and allowed for accurate digestion of the insulin precursors, and this provides a reference for Lys-C recombinant expression and potential industrial application.
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Key words:
- Lysyl endopeptidase /
- Recombinant expression /
- Renaturation /
- Purification /
- Activity analysis
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图 1 Lys-C和pre-N-pro的基因与蛋白序列
Figure 1. The gene and protein sequences of Lys-C and pre-N-pro
图 2 质粒pET28a-LysC和pET28a-preNpro的构建与验证
Figure 2. The Construction and verification of plasmids pET28a-LysC and pET28a-preNpro
图 6 Lys-C酶切胰岛素前体的HPLC图谱
Figure 6. The HPLC chromatograms of the insulin precursor digested by recombinant Lys-C
表 1 本研究使用引物
Table 1. Primers used in this study
Primers Sequence (5’ to 3’) T7lacO-F CACCATTAAGATCCGGCTGCTAAC T7lacO-R CCATGGTATATCTCCTTCTTAAAG T7lacOLysC-F AAGAAGGAGATATACCATGGGCAGCAAAGGTGTGAG T7lacOLysC-R GTTAGCAGCCGGATCTTAATGGTGGTGATGATGGTGAACTGG 28aarmpNp-F TAAGAAGGAGATATACCATGAAACGCATTTGCGGTAG pNp28ahisarm-R TTAATGGTGGTGATGATGGTGTTTTTCGCCGCTGGCGG V28aUP-F GGAAGCAGCCCAGTAGTAGG VT7DO-R CAAGACCCGTTTAGAGGCCC V28aDO-R CGATGGCCCACTACGTGAAC 
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表 3 HPLC洗脱程序
Table 3. HPLC elution procedure
Time/min A B 0 62% 38% 20 54% 46% 25 54% 46% 28 10% 90% 32 62% 38% 40 Stop Stop
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表 2 比色法测酶活的操作方法
Table 2. Method for measuring Lys-C activity by chromogenic reaction using Bz-Lys-pNA as a substrate
Reagents Control Lys-C sample A 200 μL 200 μL B 20 μL 20 μL 30℃ water bath, preheat for 5 min C 10 μL -- D -- 10 μL Mix quickly, 30℃ water bath for 30 min E 70 μL 70 μL
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表 4 Lys-C各步纯化结果
Table 4. Lys-C purification results of each step
Steps Volume/mL Protein amount/mg Recovery/% Cell lysis -- 7600 -- Washing -- 2400 -- Denaturation 130 640 -- GFC (Sephadex G25) 270 550 85.9 TFF 250 430 78.2 NCAC 40 95 22.1 UF 20 57 60.5 GFC (Sephacryl S-100) 42 48 84.8
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表 5 大肠杆菌重组表达Lys-C的产量结果
Table 5. Yield analysis of recombinant expression of Lys-C in E. coli.
Source Host Plasmid Yield Literature Achromobacter E. coli lon- pKK233-2 0.48 mg/L 1989，Ohara[21] Achromobacter E. coli JA221 pKYN200 0.3~0.4 mg/L 2002，Kentaro[8] Lysobacter E. coli JM109 pGEM-T 11 mg/L 2002，Shigeru[11] Lysobacter E. coli JM109 pJOE 24 mg/L 2016，Stressler[22] Pseudomonas E. coli BL21(DE3) pET-32a 34.4 mg/L 2016，Zhao[23] Lysobacter E. coli JM109(DE3) pET-28a 48 mg/L This study
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