Screening, Expression, and Validation of Nanobodies with FLAG Tag

doi:10.13560/j.cnki.biotech.bull.1985.2023-0291

Abstract

Abstract:

Nanobodies are a novel type of engineered antibodies with small size, high stability, and strong affinity, offering new possibilities for scientific research. The FLAG tag is a short peptide tag widely used in biological research, playing a significant role in various studies. In order to prepare nanobodies against the FLAG tag, high-affinity nanobodies were screened using yeast two-hybrid technology and the prepared FLAG nanobodies were subsequently tested for performance. Through DNA recombination technology, a bait vector containing the FLAG tag was constructed. Using yeast two-hybrid technology, nanobodies against the FLAG tag were screened from a camel-derived nanobody yeast library. Five single candidate antibody DNA sequences were screened from the yeast library. To exclude interference from the expression of protein sequences in the carrier itself on antibody screening, the possibility of non-specific hybridization was ruled out through a ‘point-to-point’ verification method. This operation confirmed that all five screened nanobodies specifically reacted with the FLAG tag. For the production of nanobodies, five nanobody prokaryotic expression vectors were constructed and expressed using the Escherichia coli system. SDS-PAGE and Western blot（WB）analysis showed that two soluble anti-FLAG tagged protein nanobodies were successfully obtained. The effects of these two nanobodies were compared with commercially available conventional FLAG tag antibodies. The results showed that both the prepared nanobodies and the commercial antibodies recognized FLAG peptides and fusion proteins with FLAG tags, and there were no significant differences in specificity, indicating that the prepared FLAG nanobodies had good application prospects. Based on yeast two-hybrid technology, FLAG tag nanobodies were successfully screened and prepared. This achievement not only enriches the types of nanobodies but also provides a new approach for the development and application of antibodies. This study provides strong support for further research on the application of FLAG tags in biological research, as well as the application of nanobodies in bioengineering.

Key words: FLAG tag, nanobody, yeast two-hybrid, screening, expression, specificity

WANG Xin-yi, WANG Xiao-qian, WANG Hong-jun, CHAO Yue-hui. Screening, Expression, and Validation of Nanobodies with FLAG Tag[J]. Biotechnology Bulletin, 2023, 39(10): 323-331.

Figures/Tables 8

Table 1 Primer sequences used in the experiment

引物名称 Primer name	序列 Sequence（5'-3'）
pGBKT7-FLAG-F	GGAGGCCGAATTCCCGGACTATAAAGACGACGACGATAAAGGGATCCGTCGACCT
pGBKT7-FLAG-R	AGGTCGACGGATCCCTTTATCGTCGTCGTCTTTATAGTCCGGGAATTCGGCCTCC
T7 promoter	AATACGACTCACTATAGGGC
3'BD	TTTTCGTTTTAAAACCTAAGAGTC
3'AD	AGATGGTGCACGATGCACAG
pCold-VHH-F	GATTACGCTCATATGGCCATGGAGGCCAGTCAGGTTCAGCTGCAGGAGTCTGGRGGAGG
pCold-VHH-R	GTTTTTCAGTATCTACGATTCATCTGCAGCTTACGCAGAAGAGACGGTGACCWGGGT
pCold-F	AAACCACTCCACTGCGTCGTCTG
pCold-R	CAGGGATCTTAGATTCTGTG

Fig. 1 Yeast two-hybrid screening of FLAG tag

Fig. 2 Sequences and structures of FLAG-tag nanobody FR1-4: Four fragment regions. CDR1-3: Three complementarity determining regions

Fig. 3 Yeast two-hybrid assay for protein-protein interaction *: pGADT7-Nanobody+pGBD T7-FLAG. **: pGADT7-Nanobody+pGBDT7 empty vector

Fig. 4 PCR detection of prokaryotic expression vector for nanobodies M: MB2000 DNA marker. 1-5: Prokaryotic expression vectors of 5 nanobodies

Fig. 5 Prokaryotic expression M: Protein marker. 1: Nano-FLAG6. 2: Nano-FLAG7. CK-: Negative controls. Red arrows: Expressed nanobodies

Fig. 6 Western blot detection M: Protein marker. 1: Nano-FLAG6. 2: Nano-FLAG7. CK-: Negative controls

Fig. 7 Western blot validation of nanobodies 1: Nano-FLAG6. 2: Nano-FLAG7. C: Commercialized FLAG monoclonal antibody. D: Schematic diagram

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