前导肽介导的SprD蛋白体外折叠研究

doi:10.13304/j.nykjdb.2023.0176

摘要/Abstract

摘要：

为提高以前体形式合成的蛋白质折叠与成熟效率，以枯草杆菌蛋白酶SprD为研究对象，通过蛋白重组表达、氨基酸定点突变、包涵体纯化结合体外复性、酶催化性能测定等方法比较各前体蛋白折叠成熟情况。结果表明，野生型前导肽介导的SprD前体蛋白可以在230 min内完成成熟，时间较长；成熟肽序列中E112A、S221A及S221C等突变导致相应前体蛋白成熟时间延长或成熟失败；而前导肽串联表达和切割位点酪氨酸缺失等措施可以将蛋白成熟时间缩短至80~160 min，成熟效率提高0.5~2.0倍，且成熟酶催化能力不受影响。前导肽序列中单个氨基酸位点改变对蛋白成熟影响较小，但对酶催化能力提升有一定帮助。因此，前导肽介导的蛋白成熟与酶催化能力的改变是2个相对独立的过程，前导肽表达方式与切割位点变化是促进蛋白体外成熟的较优方案。研究结果为加速蛋白成熟与蛋白改造提供了可参考方法。

关键词: 前导肽, SprD蛋白, 蛋白复性, 折叠, 成熟

Abstract:

To improve the folding and maturation efficiency of proteins that were first synthesized as precursors， the bacterial-derived SprD subtilisin was used as the research object and the folding and maturation of precursors were compared comprehensively by taking recombination expression of proteins， site-directed mutation of amino acids， purification of inclusion bodies， refolding of proteins in vitro anddetermination of enzyme catalytic abilities many other measures. The resluts showed that SprD precursors took about 230 min to complete the maturation under the mediation of wild-type pro-peptides. Mutations of E112A， S221A and S221C in sequences of mature peptide led to the prolongation or the failure of maturation. The tandem expression of pro-peptide and deletion of tyrosine at cleavage site between pro-peptide and mature peptide shortened the maturation time to 80~160 min and increased the maturation efficiency by 0.5~2.0 times， and the catalytic abilities of the corresponding mature enzymes were not affected. Although the single-site mutations in pro-peptide had little effects on maturation， they contributed the improvement of catalytic ability. Therefore， the maturation of proteins mediated by pro-peptides and the alternation of enzyme catalytic ability were 2 relatively independent processes and the expression modes of pro-peptide and the changes of cleavage site were suitable ways to promote protein maturation in vitro. Above results would provide a reference method for accelerating protein maturation and protein modification.

Key words: pro-peptide, SprD protein, protein renaturation, folding, maturation

中图分类号:

Q816

李丹. 前导肽介导的SprD蛋白体外折叠研究[J]. 中国农业科技导报, 2024, 26(9): 54-61.

Dan LI. Study on Folding of SprD Protein Mediated by Pro-peptide in vitro[J]. Journal of Agricultural Science and Technology, 2024, 26(9): 54-61.

图/表 8

表1 研究所用引物

Table 1 Primers used in the study

引物名称 Primer name	引物序列 Primer sequence （5’ - 3’）
pro-sprD sense	CCATGGGCGCAGGGAAATCAAACGGGGAA （Nco I）
pro-sprD anti	CTCGAGCTGAGCTGCCGCCTGTAC （Xho I）
Y-1D’ sense	GATCACGTTGCACAAGCGGCGCAGTCCGTGCCTTAC
Y-1D’ anti	GGCACGGACTGCGCCGCTTGTGCAACGTGATC
G-44D sense	GTCATTTCTGAAAAAGACGGGAAAGTGCAAAAGC
G-44D anti	GCTTTTGCACTTTCCCGTCTTTTTCAGAAATGAC
I-48V sense	CCAAGAAAAAAGATGTC GTTTCTGAAAAAGGCG
I-48V anti	CGCCTTTTTCAGAAAC GACATCTTTTTTCTTGG
E112A sense	CGGAATTGCGTGGGCGATCGCAAACAATATC
E112A anti	GCGATCGCCCACGCAATTCCGTTAATGATC
S221A sense	GTACAATGGTACG GCAATGGCATCTCCGCACG
S221A anti	GCGGAGATGC CATTGCCGTACCATTGTACG
S221C sense	GTGCGGAGATGCCAT ACACGTACCATTGTACGCGC
S221C anti	GTACAATGGTACGTGT ATGGCATCTCCGCACGT

图1 不同酶来源的前导肽序列比较

Fig. 1 Sequence alignment of pro-peptides from different enzymes

图2 野生型前体蛋白体外加工为成熟酶A：野生型前体蛋白的折叠与成熟；B~D： E112A、S221A和S221C前体蛋白的折叠与成熟

Fig. 2 Processing of wild-type precursors to mature enzymes in vitroA： Folding and maturation of wild-type precursor； B~D： Folding and maturation of E112A， S221A and S221C precursors

图3 Pro-pro-SprD前体蛋白的成熟过程A：前导肽串联表达示意图；B： Pro-pro-SprD前体蛋白的加工与成熟

Fig. 3 Maturation process of pro-pro-SprD precursorA： Schematic of tandem expression of pro-peptides； B： Processing and maturation of pro-pro-SprD precursors

图4 I-48V前体蛋白的成熟过程

Fig. 4 Maturation process of I-48V precursor

表2 成熟酶催化性能

Table 2 Catalytic properties of active enzymes

前体蛋白Precursor	突变范围 Mutation range	成熟时间 Maturation time/min	米氏常数 K_m/（μmol·L^-1）	催化常数 k_cat/（s^-1）	催化常数/米氏常数 k_cat/K_m/（s^-1·mmol^-1·L）
WT	无 No	230~240	75.69±12.00	47.02±7.00	621
I-48V	前导肽 Pro-peptide	200~210	57.68±11.00	46.53±6.00	807
G-44D	前导肽 Pro-peptide	210~220	80.45±13.00	10.55±3.00	131
Y-1D’	前导肽 Pro-peptide	80~90	66.68±10.00	45.19±5.00	678

图5 Y-1D’前体蛋白的成熟过程

Fig. 5 Maturation process of Y-1D’ precursor

图6 前体蛋白影响成熟酶活性发挥A：前体蛋白对成熟酶的抑制作用；B：前体蛋白对成熟酶的相对抑制作用

Fig. 6 Precursor proteins affect the activity of mature enzymesA： Precursors inhibited the action of active enzymes； B： Relative inhibition of precursors on enzymes

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