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BMC Biotechnol
2024 Jun 26;241:44. doi: 10.1186/s12896-024-00871-4.
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Increased stable integration efficiency in CHO cells through enhanced nuclear localization of Bxb1 serine integrase.
Huhtinen O
,
Prince S
,
Lamminmäki U
,
Salbo R
,
Kulmala A
.
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BACKGROUND: Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with mammalian glycosylation and its inherent ability to select antibodies with good biophysical properties, the restricted library size and large culture volumes remain challenges. Bxb1 serine integrase is commonly used for the stable genomic integration of antibody genes into mammalian cells, but presently lacks the efficiency required for the display of large mammalian display libraries. To increase the Bxb1 integrase-mediated stable integration efficiency, our study investigates factors that potentially affect the nuclear localization of Bxb1 integrase.
METHODS: In an attempt to enhance Bxb1 serine integrase-mediated integration efficiency, we fused various nuclear localization signals (NLS) to the N- and C-termini of the integrase. Concurrently, we co-expressed multiple proteins associated with nuclear transport to assess their impact on the stable integration efficiency of green fluorescent protein (GFP)-encoding DNA and an antibody display cassette into the genome of Chinese hamster ovary (CHO) cells containing a landing pad for Bxb1 integrase-mediated integration.
RESULTS: The nucleoplasmin NLS from Xenopus laevis, when fused to the C-terminus of Bxb1 integrase, demonstrated the highest enhancement in stable integration efficiency among the tested NLS fusions, exhibiting over a 6-fold improvement compared to Bxb1 integrase lacking an NLS fusion. Subsequent additions of extra NLS fusions to the Bxb1 integrase revealed an additional 131% enhancement in stable integration efficiency with the inclusion of two copies of C-terminal nucleoplasmin NLS fusions. Further improvement was achieved by co-expressing the Ran GTPase-activating protein (RanGAP). Finally, to validate the applicability of these findings to more complex proteins, the DNA encoding the membrane-bound clinical antibody abrilumab was stably integrated into the genome of CHO cells using Bxb1 integrase with two copies of C-terminal nucleoplasmin NLS fusions and co-expression of RanGAP. This approach demonstrated over 14-fold increase in integration efficiency compared to Bxb1 integrase lacking an NLS fusion.
CONCLUSIONS: This study demonstrates that optimizing the NLS sequence fusion for Bxb1 integrase significantly enhances the stable genomic integration efficiency. These findings provide a practical approach for constructing larger libraries in mammalian cells through the stable integration of genes into a genomic landing pad.
Fig. 1Mycobacteriophage Bxb1 serine integrase with N-terminal and C-terminal nuclear localization signal fusions (NLS). Upper construct illustrates Mycobacteriophage Bxb1 serine integrase with N-terminal NLS fusion. N-terminal NLS (yellow) and Mycobacteriophage Bxb1 serine integrase gene (blue) was separated by a GS linker (orange) and human influenza hemagglutinin (HA) epitope tag (red). Lower construct illustrates Mycobacteriophage Bxb1 serine integrase with C-terminal NLS. Amino acid sequences of the NLSs are shown below the constructs
Fig. 2 Normalized stable integration efficiencies (nSIE) of the tested Bxb1–NLS variants. (A) The graph shows the average nSIEs of Bxb1–NLS variants carrying either N-terminal (blue columns) or C-terminal (orange columns) NLS fusion. The error bars represent standard deviation of three independent experiments (except NPLC, n = 6). Asterisks denote statistically significant difference to Bxb1 without NLS (Bxb1 -NLS) (B) Bxb1–NLS variants sorted according to nSIE. Asterisks denote statistically significant difference to Bxb1–NPLC. (**) p < 0.01; (***) p < 0.001; (****) p < 0.0001
Fig. 3 Double and triple NLS variants of Bxb1 integrase. Construct on top shows the configration of the double NLS variants with NLSs at N- and C-terminus. Constructs in the middle and at the bottom illusrate the configuration of Bxb1-NPLCx2 and Bxb1-NPLCx3 variants, respectively. NLS sequences of Bxb1-NPLCx2 and Bxb1-NPLCx3 are separated by a flexible GGGGSGGGGSGS linker (white arrow)
Fig. 4 Comparison of the best single NLS variant, Bxb1-NPLC, and Bxb1–NLS variants containing two or three NLS sequences. The bars represent the average normalized stable integration efficiencies (nSIE) with error bars representing the standard deviation of three independent experiments (except NPLC, n = 6; NPLCx2, n = 9).(**) p < 0.01
Fig. 5 Co-expression of proteins involved in the translocation of proteins into the nucleus. (A) Schematic illustration of the genetic construct expressing both Bxb1–NPLCx2 and Importin α, Importin ß, RanGTP, RCC1 or RanGAP. The two proteins were separated by GS linker, furin clevage site and T2A peptide. (B) Illustration of the translocation process of proteins into the nucleus [20]. Importin α interacts with importin ß through IBB (Importin-ß-binding) domain (orange block), exposing the binding cavity of importin α, allowing the binding of NLS sequence to it. The ternary complex is then translocated into the nucleus where binding of RanGTP to importin ß causes dissociation of the ternary complex. Subsequently, importin α binds to exportin complex composed of CAS and RanGTP, and the complex is transported out from the nucleus. The exportin complex is dissociated in the cytoplasm by Ran GAP, converting Ran-GTP to RanGDP. Ran-GDP is converted back to the active Ran-GTP form in the nucleus by regulator of chromosome condensation 1 (RCC1) protein (not shown in the figure)
Fig. 6 Effect of co-expression of nuclear transport proteins. The graph shows the average normalized stable integration efficiencies (nSIE) of each Bxb1–NPLCx2-nuclear transport protein construct. The error bars represent standard deviation of three replicate transfections (except NPLCx2, n = 9; NPLCx2-RanGAP, n = 6). Asterisks denote statistically significant difference to NPLCx2. (**) p < 0.01
Fig. 7 Comparison of the normalized stable integration efficiency of Bxb1-NPLC, Bxb1-NPLCx2 with and without RanGAP co-expression using abrilumab (orange) and GFP (blue). The stable integration efficiencies are normalized to Bxb1 without NLS fusion. The error bars represent standard deviation of three replicate transfections (abrilumab) or six replicate transfections (GFP, except Bxb1-NPLCx2 n = 9). (**) p < 0.01; (***) p < 0.001
