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Updated: May 28, 2026
Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting
Published on: March 8, 2018
[Serial recombinant expression and anti-tumor activity in vitro of antibiotic peptide Alloferon-1]
1Department of Medical Microbiology and Parasitology, Zhejiang University School of Medicine, Hangzhou, China.
Objective:
To generate a recombinant expression system of repeated serial antibiotic peptide Alloferon-1 DNA segment with trypsin digestion site and to determine its anti-tumor activity in vitro.
Methods:
A 14 repeated serial DNA segment of Alloferon-1 with a lysine residual at the C-end that acts as the trypsin digestion site was constructed. pET42a vector and E.coli BL21DE3 were applied to generate the prokaryotic expression system of the repeated serial DNA segment of Alloferon-1. The yield of target recombinant product was measured by SDS-PAGE and Bio-Rad Gel image system. Ni-NTA affinity column, trypsin digestion and Sephadex G-50 column were used to purify 14 rAlloferon-1-K fusion protein and rAlloferon-1-K monomer. By using the co-cultivation of BALB/c mouse splenocyte with K562, KB or SGC tumor cells and CCK-8 detection method, the effects of rAlloferon-1-K, chemosynthetic Alloferon-1 (cAlloferon-1) and Alloferon-1-K (cAlloferon-1-K) on the growth and proliferation of tumor cells were detected.
Results:
The prokaryotic expression system E.coli BL21DE3pET42a-14 Alloferon-1-K efficiently expressed 14 rAlloferon-1-K fusion protein under inducement of IPTG,and the yield of fusion protein was approximate 30% of the total bacterial proteins. 0.1≊10 ng/ml rAlloferon-1-K remarkably increased the effect of mouse splenocytes to inhibit the growth and proliferation of K562, KB and SGC cells (P<0.05), and there was no statistically significant difference of the anti-tumor ability of rAlloferon-1-K compared to that of cAlloferon-1 or cAlloferon-1-K (P>0.05).
Conclusion:
A prokaryotic expression system of repeated serial Alloferon-1 DNA segment has been successfully constructed with high yield of rAlloferon-1-K, which maintains anti-tumor activity in vitro.
Insights
A novel recombinant expression system for the antibiotic peptide Alloferon-1 was successfully developed. This system efficiently produces a fusion protein with significant anti-tumor activity, comparable to synthetic versions.
Area of Science:
- Biotechnology
- Molecular Biology
- Immunology
Context:
- Antibiotic peptides like Alloferon-1 show promise for therapeutic applications.
- Developing efficient expression systems is crucial for large-scale production of therapeutic peptides.
- Understanding the anti-tumor mechanisms of such peptides is an active area of research.
Purpose:
- To construct a recombinant prokaryotic expression system for a repeated serial DNA segment of Alloferon-1, incorporating a trypsin digestion site.
- To purify the resulting recombinant fusion protein (rAlloferon-1-K) and its monomer.
- To evaluate the in vitro anti-tumor activity of the recombinant Alloferon-1-K against various cancer cell lines.
Summary:
- A 14-repeat serial DNA segment of Alloferon-1 with a C-terminal lysine for trypsin digestion was cloned into a pET42a vector for expression in E. coli BL21DE3.
- The recombinant fusion protein (14 rAlloferon-1-K) was expressed at high yield (~30% of total bacterial protein) and purified using Ni-NTA affinity chromatography, trypsin digestion, and Sephadex G-50 gel filtration.
- The purified rAlloferon-1-K demonstrated significant in vitro anti-tumor activity by enhancing mouse splenocyte-mediated inhibition of K562, KB, and SGC tumor cell growth and proliferation, with efficacy comparable to synthetic Alloferon-1 and its monomer.
Impact:
- Successful development of a high-yield prokaryotic expression system for a repeated serial Alloferon-1 DNA segment.
- Demonstration of potent in vitro anti-tumor activity of the recombinant Alloferon-1-K.
- Provides a foundation for further investigation and potential therapeutic development of Alloferon-1-based agents.
