Baculo Expression Vector pAc-kappa-Fc by PROGEN

Recombinant  antibody technologies have been widely used to produce various single-chain Fv or Fab antibody fragments of different specificity. The  randomized combination of cloned variable heavy and light chain  immunoglobulin gene fragments further allowed the construction of human  antibody libraries, which enable today the isolation of specific scFv or  Fab antibody fragments against particular antigens e.g. by phage  display. For many applications, however, it is required to reassemble  the variable regions of the selected antibody with immunoglobulin  constant regions to generate complete antibody molecules.

The  baculovirus expression system has been established as a reliable  system for the production of immunoglobulins (Nesbit et al., 1992;  Liang et al., 1997, 2001). Even the development of cassette vectors for  the production of human antibodies have been reported (Poul  et al., 1995a,b). However, these systems are based on a combination of  two different vectors which separately served for the expression of  light and heavy chains. The required careful and time consuming  adjustment of the two respective recombinant baculovirus titers becomes obsolete with the use of PROGEN's single baculovirus cassette  vectors with authentic IgG heavy and light chain signal sequences for  the rapid production of complete chimeric, humanized and human IgG  antibodies in recombinant baculovirus infected insect cells (Liang et  al., 2001). The vectors were specifically designed for cloning of heavy  and light chain Fv or Fab gene fragments isolated from hybridomas,  individual B cell clones as well as antibody libraries.

Vector DesignTo  achieve the expression of complete immunoglobulins, PROGEN offers a set  of baculovirus cassette vectors for the convenient insertion of heavy  and light chain Fv or Fab domain coding regions. The vectors are based  on the backbone of pAcUW51 (BD Biosciences), which contains the f1 origin of  phage DNA replication, an ampicillin resistance gene for selection in  E. coli, the two baculovirus expression promotors of polyhedrin and p10  and SV40 transcription terminators. The gene elements for IgG expression  were cloned successively into pAcUW51. The heavy chain gene cassette  preceded by the authentic IgG signal sequence from IgG1 (subgroup VHIII)  islocated under control of the polyhedrin promotor. In pAc-κ-CH3  and pAc-λ-CH3 the recognition sites of the restriction endonucleases  XhoI and NheI allow the insertion of a VH gene fragment. The entire  coding region of the human IgG1 constant domains are located downstream  thereof. The vectors pAc-κ-Fc and pAc-λ-Fc were instead designed for the  insertion of a Fab fd gene fragment using the recognition sites of the  restriction endonucleases XhoI and SpeI. The light chain gene cassette  elements, starting with the authentic signal sequence of the human kappa  chain in pAc-κ-CH3 and pAc-κ-Fc or lambda chain in pAc-λ-CH3 and  pAc-λ-Fc, were placed in opposite orientation to the heavy chain operon  under control of the p10 promotor.

The recognition sites of  the restriction endonucleases SacI and HindIII allow the insertion of a  VL gene fragment into pAc-κ-CH3 and pAc-λ-CH3. The original HindIII site  in the pAcUW51 backbone was therefore removed. The coding region of the  human constant kappa light chain is located downstream of the cloning  sites. The vectors pAc-κ-Fc and pAc-λ-Fc were designed for the insertion  of a Fab light chain gene fragment using the recognition sites of the  restriction endonucleases SacI and EcoRV (Liang et al., 2001).

Material Required

• Equipment for DNA cloning
• Basic cell culture equipment
• Insect cell line Sf9
• BaculoGold DNA, Transfection buffer A and B set (BD Biosciences)

Contents 5.0 μ;g purified plasmid DNA, 0.5 μ;g/μ;l.

Preparation of Reagents

The DNA should be diluted and transfered into E. coli cells as described  in standard protocols. Use an overnight culture of a single clone to  extract enough DNA for the following cloning procedures.

Cloning procedure The immunoglobulin heavy and light chain Fv or Fab domain coding regions  must be cloned in-frame into the selected baculovirus expression vector  (see attached graphic). To clone a VH gene fragment into pAc-κ-CH3 or  pAc-λ-CH3, the recognition sites of the restriction endonucleases XhoI  and NheI are recommended. The corresponding VL gene fragment should be  introduced by using the recognition sites of the enzymes SacI and  HindIII. To clone a Fab fd gene fragment into pAc-κ-Fc or pAc-λ-Fc, the  recognition sites of the restriction endonucleases XhoI and SpeI are  recommended. The corresponding Fab light chain gene fragment including  its terminal stop codon should be introduced by using the recognition  sites of the enzymes SacI and EcoRV. The cloning sites of the selected  Fv or Fab gene fragments have to match the corresponding amino acid  positions given by the vector, which were calculated from the first  start codon of IgG heavy and light chain mRNA coding regions. Fab genes  derived from pComb3 phagemid vector systems (Barbas et al., 1991) can be  directly cloned into pAc-κ-Fc or pAc-λ-Fc. To clone Fv or Fab genes  derived from other systems, the selected gene fragments may need to be  reamplified by PCR, thereby introducing the restriction endonucleases  sites at correct positions (Liang and Dübel., 2001). After cloning, we  recommend to check the sequence of the inserts to confirm that no  mutation occured in the open reading frames of the completed IgG heavy  and light chain genes. The vector is now ready-to-use for the generation  of recombinant baculoviruses for IgG expression in infected insect  cells.

Baculovirus transfectionWe recommend to use the baculovirus transfection kit from BD Biosciences providing linearized and modified AcNPV baculovirus DNA (BaculoGold DNA). The antibody genes supplied on PROGEN´s transfer vector are flanked by regions homologous to AcNPV. When the linearized and modified baculovirus DNA is co-transfected with PROGEN´s shuttle vector containing the Fv or Fab domain coding regions into Sf9 insect cells, recombination takes