Expression and Purification of Cell-penetrating Peptide M918 Conjugate Antibody and Study on Its Uptake Efficiency

doi:10.13560/j.cnki.biotech.bull.1985.2021-0317

Abstract

Abstract:

Application of cell-penetrating peptides in the field of drug delivery are limited in their due to the low uptake efficiency of target cells. In this study,genetic engineering methods were used to conjugate the cell penetrating peptide M918 with single-chain antibody targeting HER2,and the recombinant protein M918-scFv was expressed by Escherichia coli expression system and purified by nickel affinity chromatography. The microscale thermophoresis was applied to detect the affinity of the recombinant protein M918-scFv with the HER2 antigen. Laser confocal microscopy and flow cytometry were employed to detect the uptake efficiency of the recombinant protein. The results showed that the recombinant protein M918-scFv obtained by affinity chromatography was of high purity. Conjugating M918 did not affect the affinity of scFv and antigen. In HER2 positive cells,the uptake efficiency of the recombinant protein M918-scFv was 1.8 times that of scFv,indicating that M918-scFv exerted the penetration properties of cell-penetrating peptide M918 and presented higher uptake efficiency. Cell-penetrating peptide M918 conjugating with scFv not only demonstrated the cell-penetrating feature but also owned the targeting of scFv,thus it may provide a new targeted delivery strategy.

Key words: cell-penetrating peptide, M918, single chain antibody, targeted delivery

LI Xue, LI Jun-min, ZHANG Lei, LI Shan. Expression and Purification of Cell-penetrating Peptide M918 Conjugate Antibody and Study on Its Uptake Efficiency[J]. Biotechnology Bulletin, 2021, 37(12): 198-204.

Figures/Tables 7

Table 1 Primers used to amplify M918-scFv

Primer	Sequence（5'-3'）
F1	TGTCATATGATGGTGACCGTGCTGTTC
F2	CGGCGGGGGAGGATCTGAGCTCGCATGCGCTTGTG- GAGGAGGAGGATCTG
R1	CTGATCCTCCTCCTCCACAAGCGCATGCGAGCTCA- GATCCTCCCCCGCCG
R2	TGCAAGCTTTTTGTAGAGCTCATCCATGCCATG

Fig.1 Construction of recombinant plasmid PET-28b-M918-scFv-EGFP A:Overlap PCR amplified target gene fragment. 1:Target gene M918-scFv-EGFP. M:DNA marker. B:Screening positive clone via colony PCR. 1-6:E. coli DH5α colonies transformed with target gene. M:DNA marker. C:Plasmid profile of PET-28b-M918-scFv

Fig.2 Purification of recombinant protein M918-scFv A:Western Blot analysis of recombinant protein M918-scFv induced by different IPTG concentrations. 1:Un-induced M918-scFv sample. M:Protein marker. B:Detected and purified protein via SDS-PAGE. 1:Sample before purification. 2:Sample passing through the column. 3:10% Buffer B elution. 4:20% Buffer B elution. 5:40% Buffer B elution. 6:60% Buffer B elution. 7-8:100% Buffer B elution. M:Protein marker

Fig.3 Identification of the purified recombinant protein M918-scFv by Western Blot 1:Purified recombinant protein M918-scFv. M:Protein marker

Fig. 4 Binding curve of scFv（A）and M918-scFv（B）to HER2 antigen

Fig.5 Uptake studies of scFv and M918-scFv into different cell lines detected by flow cytometry A:Level of scFv,M918-scFv binding to SK-BR-3. B:Level of scFv,M918-scFv binding to BT474. C:Level of scFv,M918-scFv binding to MCF-7. Red:Control. Orange:MFI at 4℃. Blue:MFI at 37℃. D:Quantification of the uptake ratios of scFv and M918-scFv in SK-BR-3（a）and BT474（b）,***P < 0.001,**P < 0.01

Fig.6 Uptake studies of scFv,M918-scFv into SK-BR-3（A）,BT474（B） and MCF-7（C）cells observed by confocal laser scanning microscopy Blue:Nucleus stained with Hoechst 33342. Green:Recombinant protein. Scale bars:20 µm

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