甘蓝型油菜Kunitz蛋白酶抑制剂的异源表达与理化特征分析

doi:10.3969/j.issn.1004-1524.2022.01.14

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (1): 112-119.DOI: 10.3969/j.issn.1004-1524.2022.01.14

甘蓝型油菜Kunitz蛋白酶抑制剂的异源表达与理化特征分析

冯玮¹(), 李朋朋², 宋鹏¹^,^*()

1.聊城大学生命科学学院,山东聊城 252000
2.聊城市东昌府区农业农村局,山东聊城 252000

收稿日期:2020-09-07 出版日期:2022-01-25 发布日期:2022-02-05
通讯作者: 宋鹏
作者简介:* 宋鹏,E-mail: songpengyg@126.com
冯玮(1988—),女,山东聊城人,硕士研究生,主要从事生物活性物质研究。E-mail: fengwei01@lcu.edu.cn

Heterologous expression and physicochemical characterization of Brassica napus Kunitz protease inhibitor

FENG Wei¹(), LI Pengpeng², SONG Peng¹^,^*()

1. School of Life Sciences, Liaocheng University, Liaocheng 252000, Shandong, China
2. Bureau of Agricultural and Rural Affairs in Dongchangfu, Liaocheng, Liaocheng 252000, Shandong, China

Received:2020-09-07 Online:2022-01-25 Published:2022-02-05
Contact: SONG Peng

摘要/Abstract

摘要：

利用毕赤酵母表达系统首次克隆表达了一个油菜籽来源的Kunitz蛋白酶抑制剂基因(RTI;rti),分离纯化后对该抑制剂进行了理化特征分析。结果显示:在摇瓶发酵水平,重组RTI诱导表达水平达到628 mg·L^-1,抑制剂比活性达到69.6 TIU·mg^-1蛋白;重组RTI在30~90℃可保持70%以上抑制活性,在pH 2.0~11.0 保持80%以上抑制活性;金属离子Cu²⁺和Co²⁺,有机试剂甲醇、乙醇、丙酮和氯仿对其有抑制作用;重组RTI以非竞争性抑制方式与胰蛋白酶作用,对胰蛋白酶具有很强的、专一性抑制作用,属于典型的植物Kunitz型胰蛋白酶抑制剂。结合其良好的理化特性,重组RTI具有开发为抗虫蛋白的潜力。

关键词: 油菜籽Kunitz蛋白酶抑制剂, 毕赤酵母, 克隆表达, 理化特征

Abstract:

A Kunitz inhibitor gene (RTI;rti)from rapeseed was cloned and expressed in Pichia pastoris expression system for the first time. Its physicochemical characteristics were investigated after separation and purification. The results showed that the expression level of recombinant RTI reached 628 mg·L^-1 and its inhibitory activity reached 69.6 TIU·mg^-1 in shaking flask fermentation. The recombinant RTI maintained over 70% inhibitory activity between 30-90 ℃ and retained over 80% inhibitory activity between pH 2.0-11.0. The metal ions Cu²⁺ and Co²⁺, the organic reagents methanol, ethanol, acetone or chloroform inhibited the activity of RTI. The recombinant RTI reacted with trypsin in a non-competitive manner, and demonstrated a strong and specific inhibitory effect on trypsin. These results indicated that RTI was a typical plant Kunitz trypsin inhibitor. Combined with good physicochemical properties, RTI has the potential to be developed as insect resistant protein.

Key words: rapeseed Kunitz protease inhibitor, Pichia pastoris, cloning and expression, physicochemical characteristics

中图分类号:

S436.421

冯玮, 李朋朋, 宋鹏. 甘蓝型油菜Kunitz蛋白酶抑制剂的异源表达与理化特征分析[J]. 浙江农业学报, 2022, 34(1): 112-119.

FENG Wei, LI Pengpeng, SONG Peng. Heterologous expression and physicochemical characterization of Brassica napus Kunitz protease inhibitor[J]. Acta Agriculturae Zhejiangensis, 2022, 34(1): 112-119.

图/表 8

图1 不同来源Kunitz蛋白酶抑制剂进化树线段长度表示MEGA 5.0 计算的遗传距离;分支节点上的数字表示bootstrap百分比,小于50%数值未显示;选择建立进化树的各Kunitz蛋白酶抑制剂都已被分离鉴定,分别用各自的GenBank登录号或UniProt识别号表示;括号中标注的是Kunitz蛋白酶抑制剂的物种来源;粗体标注的是本研究油菜籽Kunitz蛋白酶抑制剂。

Fig.1 Phylogenetic tree describing the genetic distances among Kunitz protease inhibitors from different sources The line length represented the genetic distance calculated by MEGA 5.0; the numbers at nodes representedthe bootstrap values (%), values lower than 50 were not shown;all Kunitz protease inhibitors selected to establish the phylogenetic tree had been isolated and identified,each sequence was identified by its GenBank or UniProt accession number; the species source of Kunitz protease inhibitors were indicated in brackets; the Kunitz protease inhibitor from rapeseed (in this study) was labeled in bold.

表1 重组RTI的分离和纯化

Table 1 Isolation and purification of recombinant RTI

纯化步骤 Purification procedures	总蛋白 Total protein/mg	抑制剂活性 Inhibitor activity/TIU	抑制剂比活性 Specific activity of inhibitor/TIU·mg^-1	得率 Yield/%	纯化倍数 Purification fold
上清发酵液Supernatant	71±2.6 a	4938.7±204.8 a	69.6±2.8 c	100±3.4 a	1.0±0.03 c
透析、盐析Dialysis and salting out	28±0.9 b	3142.6±124.5 b	112.2±5.7 b	63.7±2.8 b	1.6±0.05 b
离子交换层析Ion exchangechromatography	1.9±0.04 c	2718.0±98.3 c	1430.5±65.9 a	55.0±1.9 c	20.6±1.10 a

图2 SDS-PAGE分析RTI的纯化情况 M,蛋白分子量标准;1,上清液;2,透析、盐析后的RTI;3,离子交换层析后纯化后的RTI。

Fig.2 SDS-PAGE analysis of the purification of RTI M, Marker; 1, Supernatant; 2, RTI after dialysis and salting out; 3, Purified RTI after ion exchange chromatography.

图3 温度对RTI的影响曲线图上没有相同小写字母表示各处理间差异显著(P<0.05)。下同。

Fig.3 Effect of temperature on RTI Different lowercase letters above the diagram represent statistically significant (P<0.05) differences among treatments. The same as below.

图4 pH对RTI的影响

Fig.4 Effect of pH on RTI

表2 金属离子和有机溶剂对RTI活性的影响

Table 2 Effects of metal ions and organic solvents on the inhibitory activity of RTI

金属离子 Metal ions	相对抑制活性 Relative inhibitory activity/%	有机溶剂 Organic solvents	相对抑制活性 Relative inhibitory activity/%
对照 Control	100±5.2 a	甲醇Methanol	87.1±1.9 c
K⁺	96.7±4.8 a	乙醇Ethanol	82.5±3.7 d
Na⁺	97.2±5.3 a	丙酮Acetone	71.8±5.3 e
Ca²⁺	95.8±6.4 a	氯仿Chloroform	62.3±2.4 f
Zn²⁺	99.3±4.1 a
Mg²⁺	97.9±5.1 a
Cu²⁺	92.4±2.8 b
Mn²⁺	96.1±6.2 a
Fe²⁺	97.3±7.1 a
Co²⁺	91.2±1.9 b

图5 RTI抑制动力学曲线 a, b和c 分别添加0、3和6 μg·mL-1抑制剂。

Fig.5 Inhibition kinetic curve of RTI a, b and c was added with 0, 3, and 6 μg·mL-1 inhibitor, respectively.

表3 RTI对不同蛋白酶的抑制作用

Table 3 Inhibitory effect of RTI on different proteases

蛋白酶Proteases	抑制率Inhibition rate/%
胰蛋白酶Trypsin	90.8±6.7 a
胰凝乳蛋白酶Chymotrypsin	38.6±2.1 b
胃蛋白酶Pepsin	0
木瓜蛋白酶Papain	0
碱性蛋白酶Alkaline protease	0

参考文献 31

[1]	廖海, 杜林方, 周嘉裕. 植物中蛋白类蛋白酶抑制剂的研究进展[J]. 天然产物研究与开发, 2002, 14(1): 80-84.
	LIAO H, DU L F, ZHOU J Y. Progress on the study of plant proteinase inhibitors[J]. Natural Product Research and Development, 2002, 14(1): 80-84.(in Chinese with English abstract)
[2]	CLEMENTE A, SONNANTE G, DOMONEY C. Bowman-Birk inhibitors from legumes and human gastrointestinal health: current status and perspectives[J]. Current Protein & Peptide Science, 2011, 12(5): 358-373.
[3]	LUIZA VILELA OLIVA M, DA SILVA FERREIRA R, GASPERAZZO FERREIRA J, et al. Structural and functional properties of kunitz proteinase inhibitors from Leguminosae: a mini review[J]. Current Protein & Peptide Science, 2011, 12(5): 348-357.
[4]	TURRA D, LORITO M. Potato type I and II proteinase inhibitors: modulating plant physiology and host resistance[J]. Current Protein & Peptide Science, 2011, 12(5): 374-385.
[5]	VOLPICELLA M, LEONI C, COSTANZA A, et al. Cystatins, serpins and other families of protease inhibitors in plants[J]. Current Protein & Peptide Science, 2011, 12(5): 386-398.
[6]	张莉, 汪东风, 张宾, 等. 豆类植物蛋白酶抑制剂研究进展[J]. 大豆科学, 2006, 25(3): 314-319.
	ZHANG L, WANG D F, ZHANG B, et al. Advances in the study of protease inhibitors from leguminous plants[J]. Soybean Science, 2006, 25(3): 314-319.(in Chinese with English abstract)
[7]	万善霞, 王婉琬, 滑静, 等. 胰蛋白酶抑制剂在不同领域的研究概况[J]. 北京农学院学报, 2003, 18(2): 152-155.
	WAN S X, WANG W W, HUA J, et al. Research status of trypsin inhibitor in different fields[J]. Journal of Beijing Agricultural College, 2003, 18(2): 152-155.(in Chinese with English abstract)
[8]	SABOTIČ J, KOS J. Microbial and fungal protease inhibitors: current and potential applications[J]. Applied Microbiology and Biotechnology, 2012, 93(4): 1351-1375. DOI URL
[9]	BENDRE A D, RAMASAMY S, SURESH C G. Analysis of Kunitz inhibitors from plants for comprehensive structural and functional insights[J]. International Journal of Biological Macromolecules, 2018, 113: 933-943. DOI URL
[10]	曲晓华, 浦冠勤. 蛋白酶抑制剂的研究与应用[J]. 蚕桑茶叶通讯, 2003(1): 19-22.
	QU X H, PU G Q. Study and application on the inhibitor of albumentenzyme[J]. Newsletter of Sericulture and Tea, 2003(1): 19-22.(in Chinese)
[11]	SATTAR R, ALI S A, KAMAL M, et al. Molecular mechanism of enzyme inhibition: prediction of the three-dimensional structure of the dimeric trypsin inhibitor from Leucaena leucocephala by homology modelling[J]. Biochemical and Biophysical Research Communications, 2004, 314(3): 755-765. DOI URL
[12]	KRAUCHENCO S, PANDO S C, MARANGONI S, et al. Crystal structure of the Kunitz (STI)-type inhibitor from Delonix regia seeds[J]. Biochemical and Biophysical Research Communications, 2003, 312(4): 1303-1308. DOI URL
[13]	HERNÁNDEZ-NISTAL J, MARTÍN I, JIMÉNEZ T, et al. Two cell wall Kunitz trypsin inhibitors in chickpea during seed germination and seedling growth[J]. Plant Physiology and Biochemistry, 2009, 47(3): 181-187. DOI URL
[14]	曾英, 桑玉英, 胡金勇, 等. 波叶青牛胆胰蛋白酶抑制剂的纯化及其性质研究[J]. 云南植物研究, 2002, 24(1): 103-108.
	ZENG Y, SANG Y Y, HU J Y, et al. Purification and characterization of a trypsin inhibitor from Tinospora crispa[J]. Acta Botanica Yunnanica, 2002, 24(1): 103-108.(in Chinese with English abstract)
[15]	魏丽冬, 王庆民, 徐同文, 等. 刺桐胰蛋白酶抑制剂的高效表达、纯化、亲和介质的制备及其在r-PA纯化中的应用[J]. 药物生物技术, 2010, 17(4): 283-286.
	WEI L D, WANG Q M, XU T W, et al. Expression, purification of Erythrina trypsin inhibitor(ETI) and its preparation, and application of ETI affinity chromatography medium[J]. Pharmaceutical Biotechnology, 2010, 17(4): 283-286.(in Chinese with English abstract)
[16]	王转花, 荆艳萍, 毛建锋. 苦荞胰蛋白酶抑制剂提取方法的研究[J]. 山西大学学报(自然科学版), 1999, 22(1): 3-5.
	WANG Z H, JING Y P, MAO J F. Study of extraction on trypsin inhibitor in Tartary buckwheat[J]. Journal of Shanxi University (Natural Science Edition), 1999, 22(1): 3-5.(in Chinese with English abstract)
[17]	王竞. 白菜型油菜种子胰蛋白酶抑制剂的分离纯化与部分性质研究[D]. 成都: 四川大学, 2005.
	WANG J. Purification and partial characteristics of a trypsin inhibitor from Brassica campestris seeds[D]. Chengdu:Sichuan University, 2005. (in Chinese with English abstract)
[18]	张燕青, 王鑫, 宋鹏, 等. 大豆胰蛋白酶抑制剂的异源表达与生化特性解析[J]. 食品工业科技, 2018, 39(24): 134-138.
	ZHANG Y Q, WANG X, SONG P, et al. Heterologous expression and biochemical characterization of soybean trypsin inhibitor[J]. Science and Technology of Food Industry, 2018, 39(24): 134-138.(in Chinese with English abstract)
[19]	ROY D N, BHAT R V. Trypsin inhibitor content in some varieties of soya bean (Glycine max L.) and sunflower seeds (Helianthus annuus L.)[J]. Journal of the Science of Food and Agriculture, 1974, 25(7): 765-769. DOI URL
[20]	TAMURA K, PETERSON D, PETERSON N, et al. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[J]. Molecular Biology and Evolution, 2011, 28(10): 2731-2739. DOI URL
[21]	MACAULEY-PATRICK S, FAZENDA M L, MCNEIL B, et al. Heterologous protein production using the Pichia pastoris expression system[J]. Yeast (Chichester, England), 2005, 22(4): 249-270. DOI URL
[22]	王荣春, 孙建华, 何述栋, 等. 胰蛋白酶抑制剂的结构与功能研究进展[J]. 食品科学, 2013, 34(9): 364-368.
	WANG R C, SUN J H, HE S D, et al. Recent advance in research on the structure and function of trypsin inhibitor[J]. Food Science, 2013, 34(9): 364-368.(in Chinese with English abstract)
[23]	蒋明, 沈涛, 徐辉碧, 等. 金属离子对蛋白质的折叠、识别、自组装及功能的影响[J]. 化学进展, 2002, 14(4): 263-272.
	JIANG M, SHEN T, XU H B, et al. The influences of metal ions on protein folding, recognition, self-assembly and biological functions[J]. Progress in Chemistry, 2002, 14(4): 263-272.(in Chinese with English abstract)
[24]	孙万儒. 有机溶剂在酶反应中的应用[J]. 微生物学通报, 1985, 12(2): 79-82.
	SUN W R. Application of organic solvents in enzyme reaction[J]. Microbiology China, 1985, 12(2): 79-82. (in Chinese)
[25]	钱民协, 黄其辰, 贾一四, 等. 有机溶剂对伴刀豆球蛋白A的晶体结构的影响[J]. 中国科学(B辑), 1998, 28(4): 289-296.
	QIAN M X, HUANG Q C, JIA Y S, et al. Effect of organic solvents on the crystal structure of concanavalin A[J]. Science in China (Series B), 1998, 28(4): 289-296.(in Chinese)
[26]	王旻. 即墨野生大豆(Glycine soja Sieb.et Zucc)的营养评价及其胰蛋白酶抑制剂的研究[D]. 青岛:中国海洋大学, 2013.
	WANG M. Study on the nutritional evaluation and trypsin inhibitor of wild soybean(Glycine soja Sieb.et Zucc) in Jimo[D]. Qingdao:Ocean University of China, 2013.(in Chinese with English abstract)
[27]	谷春梅, 宋鑫秀, 赵琳琳, 等. Kunitz型大豆胰蛋白酶抑制剂(KTI)分离纯化方法的比较[J]. 食品科技, 2014, 39(6): 234-238.
	GU C M, SONG X X, ZHAO L L, et al. Comparison of separation and purification methods of soybean Kunitz-type trypsin inhibitor(KTI)[J]. Food Science and Technology, 2014, 39(6): 234-238.(in Chinese with English abstract)
[28]	赵伟伟. 黑豆胰蛋白酶抑制剂的分离纯化及活性鉴定[D]. 太原:山西大学, 2016.
	ZHAO W W. Purification and bioactivities of trypsin inhibitor from black soybean[D]. Taiyuan: Shanxi University, 2016. (in Chinese with English abstract)
[29]	MACEDO M L R, GARCIA V A, FREIRE M D G M, et al. Characterization of a Kunitz trypsin inhibitor with a single disulfide bridge from seeds of Inga laurina(SW.) Willd[J]. Phytochemistry, 2007, 68(8): 1104-1111. DOI URL
[30]	DE MEDEIROS A F, DE SOUSA COSTA I, DE CARVALHO F M C, et al. Biochemical characterisation of a Kunitz-type inhibitor from Tamarindus indica L. seeds and its efficacy in reducing plasma leptin in an experimental model of obesity[J]. Journal of Enzyme Inhibition and Medicinal Chemistry, 2018, 33(1): 334-348. DOI URL
[31]	RAWLINGS N D, BARRETT A J, FINN R. Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors[J]. Nucleic Acids Research, 2016, 44(D1): D343-D350. DOI URL

甘蓝型油菜Kunitz蛋白酶抑制剂的异源表达与理化特征分析

Heterologous expression and physicochemical characterization of Brassica napus Kunitz protease inhibitor

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 8

参考文献 31

相关文章 7

编辑推荐

Metrics

本文评价

[1]	张高峰, 杨侃侃, 张谦, 王元红, 俞赵荣, 张学琪, 鲁智敏, 刘自敏, 李永东, 王勇. 羊口疮病毒安徽株023基因的原核表达与生物信息学分析[J]. 浙江农业学报, 2019, 31(1): 30-38.
[2]	汤先泽1，刘伟2，皮雄娥2，尹业师2，王欣2，刘新利1，*. 磷脂酰肌醇特异性磷脂酶C基因在乳酸乳球菌中的异源表达[J]. 浙江农业学报, 2016, 28(4): 640-.
[3]	刘大群;华颖. CO²胁迫对膜醭毕赤酵母细胞膜通透性的影响[J]. , 2014, 26(3): 0-786790.
[4]	赵立娜;崔言顺;任建亭;李建亮;黄兵;李玉峰;马秀丽;* . 基因C型鸭肝炎病毒VP1基因的克隆表达及其多克隆抗体的制备([J]. , 2011, 23(2): 0-262.
[5]	刘燕;庞安娜;韦强;鲍国连*;季权安;肖琛闻. Ⅰ型鸭肝炎病毒VP3基因的原核表达及其抗原性[J]. , 2011, 23(1): 66-69.
[6]	亓丽红;艾武;黄兵;刘涛;秦卓明;宋敏训*. 鸡传染性支气管炎病毒S1基因在毕赤酵母中的表达及其免疫学原性分析[J]. , 2010, 22(5): 585-589.
[7]	张存;叶伟成;王一成;袁秀芳;刘蔓雯;张燕. 猪α干扰素在毕赤酵母中的高效表达和纯化[J]. , 2010, 22(1): 0-9.