Nanobody Expression in Insect Cells-Nano Antibody Special Topic-Tekbiotech-Yeast Display Service,Phage display technology

Nanobody Expression in Insect Cells

I. Recombination and screening of baculovirus expression vectors

The genome of baculovirus is huge, and the clone of foreign genes cannot be directly inserted by enzyme cutting and ligation. It must be mediated by a transfer vector to construct a new baculovirus shuttle vector Bacmid. This vector can grow in E. coli like a plasmid, and is infectious to lepidopteran insect cells. Bacmid contains the F factor replicon (which can replicate in E. coli), the kanamycin resistance gene, and the Tn7 transposition site attTn7. In the transfer vector, the foreign gene is placed under the baculovirus promoter, and the two ends are the left and right ends of Tn7. It is used to transform the E coli strain containing Bacmid, and the auxiliary plasmid provides trans action to cause transposition, and the foreign gene is transferred to the attTn7 position of Bacmid. Insect cells transfected with this Bacmid that recombined the foreign gene can obtain 100% positive recombinant virus.

II. Nanobodies

Camelid single-domain antibodies (sabs, VHHs or Nanobodies) are antibody fragments consisting only of the variable domain of the heavy chain. These VHHs have unique structural and functional characteristics because of their small size, thermal stability and high solubility. Compared with traditional antibodies, VHHs can be manufactured in microorganisms, greatly saving cost, labor and time because VHHs lack the Fc domain containing N-linked oligosaccharides. So far, VHHs have been expressed in several production systems, from prokaryotes, yeast, fungi, insect cells, mammalian cell lines to plants.

Different forms of antibodies-Tekbiotech.png

Figure 1 Different forms of antibodies

III. Nanobodies insect cell expression

Compared with traditional insect cell fermentation, insect larvae transfected with baculovirus not only achieved higher yields and biosafety standards, but also reduced production costs and simplified the process. Lidoptera insects, such as Trichoplusia ni and Podoptera frugiperda, are common hosts for heterologous protein expression. This system also demonstrates the application of N-glycans in microbial biotechnology, pharmaceutical glycoprotein expression problems; however, by using specific insect cell lines or cells containing mannosyltransferases, mammalian-type N-glycans can be produced.

Gómez-Sebastián et al. first used the modified baculovirus expression system (IBES technology) to express VHHs. The yield of VHHs obtained from insect larvae injected with different recombinant baculovirus inocula was 257 mg/L, and 50% could be recovered after purification. The expression is shown below:

Host	Expression vector	VHH format	Antigen	Production system	Yield
Trichoplusia nilarvae	Baculoviruses	VHH	Rotavirus	Larvae	257mg/L

TekBiotech uses the pIII protein of M13 bacteriophage for VHH antibody (nanobody) surface display. Our scientists constructed a phage vector with dual tags of Flag and 6*His tag, and prepared TG1 and XL1-Blue host bacteria with a transfection efficiency of 10^8. After 2-3 transfections and amplifications, we can provide customers with a high-quality VHH antibody display library with an effective library capacity of 10^8-10^9 and a packaging capacity of 10^13-10^16/ml particles.

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