Optimization and bioactivity verification of porcine recombinant visfatin with high expression and low endotoxin content using pig liver as template

https://doi.org/10.1016/j.pep.2020.105776Get rights and content

Highlights

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    Successful construction of four different prokaryotic expression strains.

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    Preparation of porcine recombinant visfatin with low endotoxin content and high biological activity.

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    Porcine recombinant visfatin promoted the inflammatory activity of RAW264.7 cells.

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    The above results proved the biological activity of porcine recombinant visfatin.

Abstract

In order to obtain the porcine recombinant visfatin protein with high expression and low endotoxin content, the current study aims to express and verify the biological activity of the purified porcine recombinant visfatin protein. Firstly, four different expression strains were successfully constructed. Then they were simultaneously induced at 37 °C for 4 h and 16 °C for 16 h. The results showed that Visfatin-pET28a-Transetta was the best strain with high protein expression and purity at 16 °C induction for 16 h. After that, endotoxin was reduced from the recombinant visfatin until the residual endotoxin was less than one endotoxin units per milliliter (EU/mL). Finally, the purified porcine recombinant visfatin protein was incubated with RAW264.7 cells. The results of cell counting kit-8 (CCK-8) showed the survival rate of the cells first increased and then decreased with the increase in visfatin concentration. When the concentration of visfatin was 700 ng/mL, the survival rate of the cells was the highest. Thereafter, control (PBS), Visfatin and Visfatin + PolymyxinB (Ploy.B) groups were incubated with the RAW264.7 cells for 6 h. Real-time quantitative polymerase chain reaction (RT-qPCR) and Enzyme Linked Immuno-Sorbent Assay (ELISA) results showed that, as compared to the control group, the expressions of interleukin (IL)-1β, tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1 in Visfatin group were significantly increased (P < 0.05). However, there was no significant difference between the Visfatin and Visfatin + Poly.B groups, indicating that porcine recombinant visfatin protein promoted the inflammatory activity of RAW264.7 cells while the residual endotoxin did not play a role, suggesting biological activity of porcine recombinant visfatin protein.

Introduction

Visfatin, a new adipocytokine, is also known as nicotinamide mononucleotide adenylyltransferase (NAMPT) or pre-B-cell colony-enhancing factor (PBEF) [1]. It is highly expressed in visceral adipose tissue [2], involved in DNA replication, apoptosis, repair and growth in different kind of cells [3]. Visfatin is a visceral fat cytokine that exerts similar effects to that of insulin [4]. As an adipocytokine, visfatin affects the immune function and inflammatory reaction [5]. Furthermore, visfatin plays an important role in insulin resistance [6], appetite promotion [7] and cancer [8]. These accumulating scientific reports emphasize the important role of visfatin in biological processes.

The structure of visfatin protein lacks typical signal peptide sequence [9]. Due to the existence of alternative splicing, visfatin encodes transcription products of various sizes [10]. There are multiple kinase phosphorylation sites on recombinant product sequences, which are involved in the biological effects of visfatin [11], mainly in the form of dimers [12]. The coding sequence of the NAMPT gene is highly conserved evolutionarily indicating its critical significance to different biological functions [13]. Tissue expression profiling of porcine visfatin gene revealed its expression in multiple porcine tissues. Chen cloned the porcine visfatin gene and studied its characteristics in three variants [10]. There are also other studies on the cloning and expression of visfatin gene, however the expression level and purity of the protein was not high [14].

Therefore, in the present study, total RNA of pig liver was used as a template and a large amount of highly purified and biologically active porcine recombinant visfatin protein was successfully obtained through prokaryotic expression and biological activity verification methods, which provided the basis for in-depth research of recombinant visfatin in the porcine field.

Section snippets

Gene cloning and identification

According to the sequence of coding region in porcine visfatin gene transcript 1 (DQ 020218 NM_01031793) available at GenBank, a pair of specific primers containing BamH Ⅰ and Xho Ⅰ restriction sites was designed; Forward (5′-CGGGATCCATGAATGCTGCGGCAGAAGCCGAATTCAAC -3′), Reverse (5′-CCGCTCGAGTTACTAATGAGGTGCTGCTTCCAGTTCAATATTCAGCTGTGCG-3′).

We extracted the total RNA from pig liver, converted it into cDNA and then amplified the target gene using this cDNA as template with the specific primers by

Visfatin gene amplification

Specific primers were used to amplify the visfatin gene by PCR. After agarose gel electrophoresis of the product, a specific band of about 1.5 kb was observed (Fig. 1a), which was consistent with the expected size (1476 bp).

Nucleic acid and amino acid sequence analysis

The forward and reverse sequencing results of the pEASY-T1-visfatin plasmid obtained from Shanghai Bio-Biotechnology Co., Ltd. were spliced to obtain the complete porcine visfatin gene-coding region (CDS) sequence. The sequence was compared with the GenBank sequence

Discussion

The Escherichia coli (E. coli) expression system has the advantages of clear genetic background, simple molecular manipulation, short culture period, with widely used host for expression analysis of recombinant proteins [15]. The target gene cloning into the pET series vector, can be controlled by the strong T7 promoter [16]. After sufficient induction, it can express more than 50% of the total protein in just a few hours [17,18]. Poly-Histidine tags or His tags are the most commonly used

Conclusion

In this study, we successfully constructed four different prokaryotic expression strains and prepared a large number of porcine recombinant visfatin protein with low endotoxin content and biological activity. Furthermore, the recombinant visfatin protein promoted the inflammatory activity of RAW264.7 cells while the residual endotoxin did not play any role, suggesting biological activity of porcine recombinant visfatin protein.

Author statement

None of the authors has any challenging conflicts of interests.

Data availability

The nucleotide sequence data has been deposited in GenBank under BankIt accession number MW052047.

Declaration of competing interest

The authors declare that they have no conflicts of interest.

Acknowledgments

National Natural Science Fund Project of China (No.31772687); Fundamental Research Funds for the Central Universities (No.2662015PY063) and National Natural Science Fund Project of China (No. 31101776).

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