Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (17): 3461-3478.doi: 10.3864/j.issn.0578-1752.2023.17.018

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

Prokaryotic Expression and Metal Binding Characterization of Metallothionein 1A and 2A of Sus scrofa

YANG HuiZhen1(), YANG Huan1, WU ZiXuan1, FAN KuoHai2, YIN Wei1, SUN PanPan1, ZHONG Jia1, SUN Na1, LI HongQuan1()   

  1. 1Shanxi Key Laboratory for Modernization of TCVM/College of Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, Shanxi
    2Shanxi Key Laboratory for Modernization of TCVM/Animal Experimental Center, Shanxi Agricultural University, Taigu 030801, Shanxi
  • Received:2022-08-26 Accepted:2023-03-28 Online:2023-09-01 Published:2023-09-08
  • Contact: LI HongQuan

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Abstract:

【Objective】 The study mainly made the bioinformatics analysis, carried out prokaryotic expression, and explored the metal binding characterization of Sus scrofa metallothionein-1A (SsMT-1A) and metallothionein-2A (SsMT-2A) with Zn(Ⅱ) and Cu(Ⅰ), so as to provide a theoretical basis for the study of the mechanism of action of adding Zn and Cu in diet inducing porcine metallothionein expression, thereby regulating the Zn and Cu homeostasis in vivo, and then promoting the porcine production performance. 【Method】 Firstly, the porcine gene sequences of SsMT-1A and SsMT-2A were obtained from NCBI. Afterwards, their protein sequence characteristics were analyzed by ClustalX2 software and ExPASy database, and then the phylogenetic tree of MT protein molecular was constructed between them and other species using the Mega-X. Secondly, the prokaryotic expression vector pET-28a-SUMO-SsMT-1A/SsMT-2A were constructed and verified by PCR, double digestion, and gene sequencing. Subsequently, the recombinant plasmid was transformed into BL21(DE3) plysS and the expression of SsMT-1A and SsMT-2A was induced using the IPTG. Then, the SUMO-SsMT-1A/SsMT-2A recombinant protein were purified by Ni-NTA affinity chromatography and Superdex-75 column, and were analyzed by Western-blot. In the end, the properties of SsMT-1A and SsMT-2A binding Zn(Ⅱ) and Cu(Ⅰ) were surveyed by the tolerance analysis of Escherichia coli containing SsMT-1A and SsMT-2A genes, circular dichroism (CD), matrix assisted laser analytic ionization time of flight mass spectrometry (MALDI-TOF-MS), and isothermal micrometer calorimetry (ITC). 【Result】 Bioinformatics analysis showed that the protein sequences of SsMT-1A and SsMT-2A were highly homologous, and their cysteine (Cys) content and arrangement motifs were perfectly consistent, and there existed only 8 amino acid residues discrepancy. Through the prokaryotic expression, purification, and SUMO enzyme digestion, the SsMT-1A/SsMT-2A fusion protein were successfully obtained. The tolerance analysis of metal demonstrated that compared with SsMT-2A, E. coli containing SsMT-1A genes had the stronger resistance to Zn and Cu. The CD spectrum illustrated that SsMT-1A and SsMT-2A could combine with Zn(Ⅱ) and Cu(Ⅰ) and both of them exhibited the preference with Zn(Ⅱ) coordination. But SsMT-1A exhibited the stronger binding ability with Zn(Ⅱ) than SsMT-2A. MALDI-TOF-MS results showed that the experimental molecular weights of apo-SsMT-1A and apo-SsMT-2A were respective 6 047.5 Da and 6 048 Da, and the stability order of both of them was and Cu(Ⅰ)> Zn(Ⅱ). ITC results showed that the affinity constants of SsMT-1A and SsMT-2A coordinating Cu(Ⅰ) were higher than that of Zn(Ⅱ), and the stoichiometries of both of them binding Cu(Ⅰ) were 7, while the stoichiometry of SsMT-1A binding Zn(Ⅱ) was 2. 【Conclusion】 Although SsMT-1A and SsMT-2A possessed the high homology, the difference of 8 amino acid residues determined their different binding feature with Zn(Ⅱ) and Cu(Ⅰ). Although SsMT-1A and SsMT-2A shared a highly consistent binding behavior of Zn(Ⅱ) and Cu(Ⅰ), their characteristics of binding Zn(Ⅱ) and Cu(Ⅰ) was slightly different. Thus, they should not be considered as completely physiological equivalent molecules. According to the relationship of structure and function of MT, SsMT-1A might play a role in detoxification of heavy metal ions and SsMT-2A might mainly regulate Zn homeostasis. The study laid a foundation for further elucidating the role of SsMT-1A and SsMT-2A in regulating metal ion homeostasis to promote pig production performance.

Key words: Sus scrofa, metallothionein-1A, metallothionein-2A, bioinformatics analysis, prokaryotic expression, metal binding characteristic

Fig. 1

Protein sequence alignment of SsMT-1A and SsMT- 2A"

Table 1

Amino acid composition of SsMT-1A and SsMT-2A"

氨基酸
Amino acid		 SsMT-1A SsMT-2A 氨基酸
Amino acid		 SsMT-1A SsMT-2A
数量
Number		 占比
Proportion (%)		 数量
Number		 占比
Proportion (%)		 数量
Number		 占比
Proportion (%)		 数量
Number		 占比
Proportion (%)
Ala（A） 7 11.5 7 11.5 Lys（K） 5 8.2 8 13.1
Arg（R） 2 3.3 0 0.0 Met（M） 1 1.6 1 1.6
Asn（N） 1 1.6 1 1.6 Phe（F） 0 0.0 0 0.0
Asp（D） 2 3.3 3 4.9 Pro（P） 3 4.9 2 3.3
Cys（C） 20 32.8 20 32.8 Ser（S） 9 14.8 8 13.1
Gln（Q） 1 1.6 1 1.6 Thr（T） 3 4.9 2 3.3
Glu（E） 0 0.0 0 0.0 Trp（W） 0 0.0 0 0.0
Gly（G） 6 9.8 6 9.8 Tyr（Y） 0 0.0 0 0.0
His（H） 0 0.0 0 0.0 Val（V） 0 0.0 1 1.6
Ile（I） 1 1.6 1 1.6 Pyl（O） 0 0.0 0 0.0
Leu（L） 0 0.0 0 0.0 Sec（U） 0 0.0 0 0.0

Table 2

Physicochemical properties of SsMT-1A and SsMT-2A"

蛋白质
Protein		 氨基酸数
Amino acid
number		 理论分子
Theoretical molecular weight (Da)		 等电点
Isoelectric
point		 不稳定系数
Instability index
(%)		 正电荷
Positive
charge		 负电荷
Negative
charge
SsMT-1A 61 5944 8.39 75.94 7 2
SsMT-2A 61 5945 8.38 60.23 8 3

Fig. 2

Phylogenic tree of between SsMT-1A and SsMT-2A and other species MT"

Fig. 3

Identification of TOP 10 E. coli containing pET-28a- SUMO-SsMT-1A/SsMT-2A vector by PCR a: PCR product of pET-28a-SUMO-SsMT-1A plasmid; b: PCR product of pET-28a-SUMO-SsMT-2A plasmids; in the a, M: 2000 DNA Ladder; Line 1: SsMT-1A gene fragment; in the b, M: 2000 DNA Ladder; Line 1: SsMT-2A gene fragment"

Fig. 4

Identification of pET-28a-SUMO-SsMT-1A/SsMT-2A plasmids by double restriction digestion M: 5000 DNA Ladder; Line 1: Identification of pET-28a-SUMO-SsMT-1A plasmid by double restriction digestion; Line 2: Identification of pET-28a-SUMO-SsMT-2A plasmid by double restriction digestion"

Fig. 5

SDS-PAGE identification of SUMO-SsMT-1A and SUMO-SsMT-2A M: Marker; Line 1: bacterial protein containing the pET-28a vector; Line 2: bacterial protein containing the pET-28a-SUMO vector; Line 3: bacterial protein containing the pET-28a-SUMO-SsMT-1A vector; Line 4: bacterial protein containing the pET-28a-SUMO-SsMT-2A vector; Line 5: the supernatant of bacterial protein containing the pET-28a-SUMO-SsMT-1A vector; Line 6: the supernatant of bacterial protein containing the pET-28a- SUMO-SsMT-2A vector"

Fig. 6

Western blot identification of SUMO-SsMT-1A and SUMO-SsMT-2A M: Marker; Line 1: the supernatant of bacterial protein containing the pET-28a-SUMO-SsMT-1A vector; Line 2: the supernatant of bacterial protein containing the pET-28a-SUMO-SsMT-2A vector"

Fig. 7

Gradient elution of Ni-NTA column purification of SUMO-SsMT-1A (left) and SUMO-SsMT-2A (right) Gradient elution of Ni-NTA column purification of SUMO-SsMT-1A and SUMO-SsMT-2A; M: Marker; Line 1: the supernatant of bacterial protein containing the pET-28a-SUMO-SsMT-1A\ SsMT-2A vector; Line 2: the flowing liquid; Line 3: the eluting liquid containing 10 mmol·L-1 imidazole; Line 4: the eluting liquid containing 25 mmol·L-1 imidazole; Line 5: the eluting liquid containing 50 mmol·L-1 imidazole; Line 6: the eluting liquid containing 100 mmol·L-1 imidazole; Line 7: the eluting liquid containing 200 mmol·L-1 imidazole; Line 8: the eluting liquid containing 300 mmol·L-1 imidazole; Line 9: the eluting liquid containing 400 mmol·L-1 imidazole"

Fig. 8

Purification of SUMO-SsMT-1A and SUMO-SsMT-2A by affinity chromatography M: Marker; Line 1 and 5: the supernatants of bacterial protein containing the pET-28a-SUMO-SsMT-1A\ SsMT-2A vector; Line 2 and 6: the flowing liquid; Line 3 and 7: the eluting liquid containing 50 mmol·L-1 imidazole; Line 4 and 8: the eluting liquid containing 200 mmol·L-1 imidazole"

Fig. 9

Purification of SUMO-SsMT-1 and SUMO-SsMT-2A by Superdex-75 column a and b：the purification of SUMO-SsMT-1A and SUMO-SsMT-2A by Superdex-75 column；In Fig.9a, Line 1-9: the protein eluate in different collecting tubes；In Fig.9b, Line 1-8: the protein eluate in different collecting tubes"

Fig. 10

Removal of SUMO label of SUMO-SsMT-1A and SUMO-SsMT-2A by SUMO enzyme M：Marker；Line 1 and 3: SUMO-SsMT-1A and SUMO-SsMT-2A undigested by SUMO enzyme; Line 2 and 4: SUMO-SsMT-1A and SUMO-SsMT-2A digested by SUMO enzyme"

Effects of different concentrations of CuSO4 and ZnSO4 on the growth of E. coli containing SsMT-1A and SsMT-2A a and e: blank group; b, c, and d: 0.75, 1.5和3 mmol·L-1 CuSO4 treatment group; f, g, and h: 0.25, 0.5和1 mmol·L-1 ZnSO4 treatment group"

Fig. 12

Effects of different concentrations of CuSO4 and ZnSO4 on the growth curve of E. coli containing SsMT-1A and SsMT-2A a, b, c: growth curves of E. coli containing SsMT-1A and SsMT-2A in the medium containing different concentrations of CuSO4; d, e, f: growth curves of E. coli containing SsMT-1A and SsMT-2A in the medium containing different concentrations of ZnSO4"

Fig. 13

UV and CD spectrum of Zn-SsMT-1A, Zn-SsMT-2A, Cu-SsMT-1A and Cu-SsMT-2A a, b, c, and d: UV spectrum of Zn-SsMT-1A, Zn-SsMT-2A, Cu-SsMT-1A, and Cu-SsMT-2A; e, f, g, and h: CD spectrum of Zn-SsMT-1A, Zn-SsMT-2A, Cu-SsMT-1A, and Cu-SsMT-2A"

Fig. 14

MALDI-TOF-MS spectrum of Zn-SsMT-1A, Zn-SsMT-2A, Cu-SsMT-1A, and Cu-SsMT-2A at pH 2.4 a and b: MALDI-TOF-MS spectrum of apo-SsMT-1A and apo-SsMT-2A; c and d: MALDI-TOF-MS spectrum of Zn-SsMT-1A and Zn-SsMT-2A at pH 2.4; e and f: MALDI-TOF-MS spectrum of Cu-SsMT-1A and Cu-SsMT-2A at pH 2.4"

Table 3

Thermodynamic parameter of ZnSO4 and CuSO4 titration of apo-SsMT-1A and apo-SsMT-2A"

蛋白质
Protein		 化学计量数
Stoichiometry		 结合常数
Binding constant (mol-1)		 焓变
Enthalpy change (joules·mol-1)		 熵变
Entropy change (joules·mol-1·deg-1)
Zn-SsMT-1A 2 （1.11±0.52）×104 -9.919×105±1.35×107 -3.23×103
Zn-SsMT-2A ─ ─ ─ ─
Cu-SsMT-1A 7 （1.15±2.4）×105 -1.631×106±4.69×107 -5.37×103
Cu-SsMT-2A 7 （1.11±1.7）×105 -1.473×106±3.05×107 -4.85×103

Fig. 15

ITC chromatograms of ZnSO4 and CuSO4 titrating apo-SsMT-1A and apo-SsMT-2A a and b: ITC chromatograms of ZnSO4 titrating apo-SsMT-1A and apo-SsMT-2A; c and d: ITC chromatograms of CuSO4 titrating apo-SsMT-1A and apo-SsMT-2A"

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