Prokaryotic Expression, Purification and Functional Verification of <i>ASAT1</i> Gene of <i>Nicotiana tabacum</i>

doi:10.13523/j.cb.2210002

China Biotechnology

2023, Vol. 43

Issue (4): 30-40 DOI: 10.13523/j.cb.2210002

Prokaryotic Expression, Purification and Functional Verification of ASAT1 Gene of Nicotiana tabacum

HAN Li^1,^**(

),WANG Li-jiao¹,XIAO Cheng-zhi¹,DONG Zi-qiang¹,DU Yue¹,HE Pei-xin^1,²

1. School of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou 450000, China
2. Collaborative Innovation Center for Food Production and Safety, Zhengzhou University of Light Industry, Zhengzhou 450000, China

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Abstract

Objective: It is reported that NtASAT1 (Nicotiana tabacum acylsugar acyltransferase) from tobacco can transform sucrose and short branched chain fatty acids to sucrose monoester. Thus, we tried to use the prokaryotic expression system of Escherichia coli to analyze the expression and purification condition of NtASAT1 and further verify the function of purified NtASAT1. Methods: First, the physical and chemical properties, secondary structure and homology of tobacco NtASAT1 were analyzed by bioinformatics software. Then, the gene NtASAT1 was cloned from the cDNA of tobacco glandular hairs and constructed into the expression vector so as to study its expression in BL21 (DE 3). Finally, NtASAT1 was purified by nickel column and the activity of purified target protein was then analyzed by enzyme reaction. The product of the enzymatic reaction was analyzed by liquid chromatography-mass spectrometry (LC-MS). Results: NtASAT1, which was truncated by 93 amino acids at the C-terminal, could be expressed in BL21 (DE3). Most of the expressed protein existed in an insoluble state and different concentrations of inducer, induction time and induction temperature had no obvious effect on the soluble expression of the protein trNtASAT1. The target protein was purified by nickel column and after enzyme reaction by adding substrate, the product sucrose monoester could be detected by LC-MS. Conclusion: The recombinant protein trNtASAT1 was cloned and purified and the enzymatic product sucrose monoester was detected by LC-MS, which proved that the purified NtASAT1 was functional. This study laid the foundation for purification and further application of enzyme ASAT in industry.

Key words： Nicotiana tabacum Acylsugar acyltransferase Expression Purification

Received: 07 October 2022 Published: 04 May 2023

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	Li HAN
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Cite this article:

HAN Li, WANG Li-jiao, XIAO Cheng-zhi, DONG Zi-qiang, DU Yue, HE Pei-xin. Prokaryotic Expression, Purification and Functional Verification of ASAT1 Gene of Nicotiana tabacum. China Biotechnology, 2023, 43(4): 30-40.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2210002 OR https://manu60.magtech.com.cn/biotech/Y2023/V43/I4/30

Fig.1 Secondary structure of NtASAT1 protein Blue, purple, red and green represent α-spiral, random crimp, extension chain β-corner, respectively; The horizontal axis represents the number of amino acids

Table 1 Homology analysis of NtASAT1 protein

Fig.2 Evolutionary tree analysis of NtASAT1 protein Sequence analysis was performed using MEGA7, the sequence alignment of ASAT from Solanum lycopersicum, Nicotiana tabacum, Petunia axillaris, Salpiglossis sinuate and wild tomato species and target species are marked in red. Phylogenetic trees were constructed using the Neighbor-Joining method and the Jones-Taylor-Thornton model. A bootstrap test of 1 000 replicates was used to assess the reliability of the phylogeny

Fig.3 Cloning of NtASAT1 gene and identification of expression vector (a) Cloning of NtASAT1 gene; M: Molecular weight standard of nucleic acid; 1: NtASAT1 cloning results (b) Colony PCR results; M: Molecular weight standard of nucleic acid; 1-4: Colony PCR identification results (c) Double enzyme digestion identification results; M: Molecular weight standard of nucleic acid; 1-4: Double digestion identification results

Fig.4 Expression of NtASAT1 protein (a) Expression of intact protein NtASAT1. M: Protein marker; 1: NtASAT1 [BL21 (DE3)] whole cell without inducer; 2: NtASAT1 [BL21 (DE3)] whole cell was added (b) The expression of truncated NtASAT1, where trNtASAT1 indicated by the arrow is the target protein. M: Protein marker; 1: The whole cell without inducer truntNtASAT1 [BL21 (DE3)]; 2: Add inducer truntNtASAT1 [BL21 (DE3)] to the whole cell (c) Amino acid sequence alignment analysis of acyl glycosyl acyltransferases from different sources and red arrow represents truncated site

Fig.5 Effects of different inducer concentrations, induction time and induction temperature on the expression of recombinant protein (a) The effect of IPTG concentration on the expression of recombinant protein, the arrow points to the target protein. M: Protein molecular weight standard; 1~2, 3~4, 5~6, 7~8 were induced by IPTG of 0.05 mmol/L, 0.1 mmol/L, 0.2 mmol/L, 0.5 mmol/L, respectively; S: Supernatant part after wall breaking; C: Sedimentation part after wall breaking (b) The effect of induction temperature on the expression of recombinant protein, the arrow points to the target protein. M: Protein molecular weight standard; 1~2, 3~4, 5~6, 7~8 were induced at 16℃, 20℃, 28℃, 37℃, respectively; S: Supernatant part after wall breaking; C: Sedimentation part after wall breaking (c) The effect of induction time on the expression of recombinant protein, the arrow refers to the target protein. M: Protein molecular weight standard; 1~2, 3~4, 5~6, 7~8 were induced for 4 h, 8 h, 12 h, 24 h, respectively; S: Supernatant part after wall breaking; C: Sedimentation part after wall breaking

Fig.6 Purification of recombinant protein M: Protein marker; 1: Whole protein; 2: Flow through; 3: Elution component (10 mmol/L imidazole); 4: Elution component (50 mmol/L imidazole); 5: Elution component (100 mmol/L imidazole); 6: Elution component (200 mmol/L imidazole); 7: Elution component (500 mmol/L imidazole)

Fig.7 Validation of trNtASAT1 enzyme activity (a) The recombinant trNtASAT1 produced S1:5 in vitro with sucrose as acyl receptor and iC5 CoA as acyl donor; Negative ion mode chromatogram with charge ratio of reactant 471.2 (b) The mass charge ratio of the product is 471.2, and the collision voltage is 20 V


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