Expression， Purification and Preliminary Identification of Recombinant Human Interleukin-27

doi:10.13560/j.cnki.biotech.bull.1985.2026-0250

Abstract

Abstract:

Objective Recombinant human interleukin-27 (rh IL-27) single-chain fusion protein tends to form inclusion bodies when heterologously expressed in Escherichia coli. In this study, we aimed to achieve high-ratio soluble expression, purification, and preliminary characterization of the protein by employing a synergistic strategy that combined the screening of solubilization tags with the co-expression of molecular chaperones. Method The synthesized human IL-27 single-chain fusion gene was cloned into the pET-32a(+) vector. High-solubility expression schemes were obtained by systematically screening six solubilization tags (XXA, SUMO, GST, NusA, TrxA, and MBP) and five sets of molecular chaperone co-expression systems. The target protein was purified via nickel affinity chromatography and identified using SDS-PAGE and Western blot. After endotoxin removal, ELISA and CCK-8 assays were used to detect the binding affinity of rh XXA-IL-27 with antibodies and its inhibitory effect on the proliferation of human breast cancer MCF-7 cells. Result Expression vectors for the IL-27 fusion gene and six solubilization tags were constructed. Screening results indicated that the XXA tag provided the best solubilization effect, with the soluble expression level of its fusion protein reaching 64.91%. Western blot analysis confirmed the specificity of the target protein. Upon further introduction of the molecular chaperone pGro7 for co-expression with the XXA tag, the maximum expression of rh XXA-IL-27 reached 46.4 mg/L, and the target protein was obtained at high concentration and purity after nickel column purification. In vitro activity assays demonstrated that treatment with 30 μg/mL rh XXA-IL-27 for 48 h significantly inhibited the proliferation of MCF-7 cells. Conclusion The synergistic strategy combining solubilization tags (especially the XXA tag) with the molecular chaperone pGro7 successfully addressed the challenge of inclusion body formation for the single-chain fusion protein IL-27 expressed in E. coli. This approach realized the efficient soluble expression and purification of the XXA-IL-27 fusion protein, yielding XXA-IL-27 with potent inhibitory activity against MCF-7 cell proliferation.

Key words: human interleukin-27, prokaryotic expression, solubility-enhancing tags, molecular chaperones, MCF-7 cells, cell proliferation inhibition

REN Yan-xiao, WEN Sai, TAN Tian-wei. Expression， Purification and Preliminary Identification of Recombinant Human Interleukin-27[J]. Biotechnology Bulletin, 2026, 42(11): 318-327.

Figures/Tables 13

Fig. 1 Schematic diagram of vector construction for pET-32a-IL-27

Fig. 2 Schematic diagram of constructing vector with different tags

Table 1 Types of chaperone proteins encoded by each plasmid and inducers

No.	质粒 Plasmid	伴侣蛋白质 Chaperone protein	启动子 Promoter	诱导物 Inducer	抗性标记 Selection marker
1	pG-KJE8	dnaK-dnaJ-grpE groES-groEL	araB Pzt-1	L-Arabinose Tetracycline	Cm
2	pGro7	groES-groEL	araB	L-Arabinose	Cm
3	pKJE7	dnaK-dnaJ-grpE	araB	L-Arabinose	Cm
4	pG-Tf2	groES-groEL-tig	Pzt-1	Tetracycline	Cm
5	pTf16	tig	araB	L-Arabinose	Cm

Fig. 3 SDS-PAGE electrophoresis analysis of various protein expression form1: pET-32a (+) uninduced; 2: pET-32a (+) whole cells; 3: pET-32a (+) intracellular supernatant; 4: pET-32a (+) intracellular pellet (the band at approximately 20 kD corresponds to the tag polypeptide expressed by the empty vector); 5: IL-27 uninduced; 6: IL-27 whole cells; 7: IL-27 intracellular supernatant; 8: IL-27 intracellular pellet; M: protein marker

Fig. 4 SDS-PAGE analysis of the effects of different solubility tags on the soluble expression of rh IL-27M: Protein marker; 1: IL-27 intracellular pellet; 2: IL-27 intracellular supernatant (54 kD); 3: XXA-IL-27 intracellular pellet; 4: XXA-IL-27 intracellular supernatant (71 kD); 5: SUMO-IL-27 intracellular pellet; 6: SUMO-IL-27 intracellular supernatant (66 kD); 7: MBP-IL-27 intracellular pellet; 8: MBP-IL-27 intracellular supernatant (91 kD); 9: GST-IL-27 intracellular pellet; 10: GST-IL-27 intracellular supernatant (76 kD); 11: NusA-IL-27 intracellular pellet; 12: NusA-IL-27 intracellular supernatant (105 kD); 13: TrxA-IL-27 intracellular pellet; 14: TrxA-IL-27 intracellular supernatant (66 kD)

Fig. 5 Densitometric analysis of rh IL-27 solubility (A) and ELISA quantification of total expression levels (B) with different solubility-enhancing tagsDifferent letters indicate significant differences among groups (P<0.05). The same below

Fig. 6 Western blot identification of expressed productsA: Anti-His-tag antibody; B: anti-IL-27 polyclonal antibody; 1: XXA-IL-27 (71 kD); 2: SUMO-IL-27 (66 kD); 3: GST-IL-27 (76 kD); 4: NusA-IL-27 (105 kD); 5: TrxA-IL-27 (66 kD); 6: MBP-IL-27 (91 kD); 7: IL-27 (54 kD); M: protein marker

Fig. 7 SDS-PAGE analysis of the effects of chaperone co-expression on XXA-IL-27 solubilityM: Protein marker; 1: XXA-IL-27 (pKJE7) intracellular pellet; 2: XXA-IL-27 (pTf16) intracellular pellet; 3: XXA-IL-27 (pGro7) intracellular pellet; 4: XXA-IL-27 (pG-KJE8) intracellular pellet; 5: XXA-IL-27 (pG-Tf2) intracellular pellet; 6: XXA-IL-27 (pKJE7) intracellular supernatant; 7: XXA-IL-27 (pTf16) intracellular supernatant; 8: XXA-IL-27 (pGro7) intracellular supernatant; 9: XXA-IL-27 (pG-KJE8) intracellular supernatant; 10: XXA-IL-27 (pG-Tf2) intracellular supernatant

Fig. 8 Densitometric analysis of XXA-IL-27 solubility (A) and ELISA quantification of total expression levels (B) upon co-expressed with different chaperones

Fig. 9 SDS-PAGE analysis of rh XXA-IL-27 purificationM: Protein marker; 1: flow-through; 2: 20 mmol/L imidazole; 3: 30 mmol/L imidazole; 4: 75 mmol/L imidazole; 5: 150 mmol/L imidazole; 6: 300 mmol/L imidazole; 7: 500 mmol/L imidazole (unconcentrated eluate)

Fig. 10 SDS-PAGE (A) and Western blot (B) analysis of TEV cleavageM: Protein marker; (A) 1: TEV protease; 2: XXA-IL-27 before cleavage; 3: XXA-IL-27 after cleavage; (B) 1: XXA-IL before cleavage; 2: XXA-IL after cleavage

Fig. 11 Effect of XXA-IL-27 treatment for 48 h on the morphology of MCF-7 cellsA: Control group; B: 5 μg/mL treatment group; C: 30 μg/mL treatment group

Fig. 12 Effect of XXA-IL-27 on MCF-7 cell viability and corresponding IC50 assessmentA: Bar chart of cell viability; B: dose-response profile and IC50 fitting curve

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