Heterologous expression and physicochemical characterization of Brassica napus Kunitz protease inhibitor

doi:10.3969/j.issn.1004-1524.2022.01.14

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (1): 112-119.DOI: 10.3969/j.issn.1004-1524.2022.01.14

• Plant Protection • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S436.421

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.

Figures/Tables 8

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.

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

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.

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.

Fig.4 Effect of pH on RTI

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

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

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

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Heterologous expression and physicochemical characterization of Brassica napus Kunitz protease inhibitor

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