| 1. |
- Faber, Catherine, et al.
(författare)
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Three-dimensional structure of a human Fab with high affinity for tetanus toxoid
- 1998
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Ingår i: Immunotechnology. - 1380-2933. ; 3:4, s. 253-270
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Tidskriftsartikel (refereegranskat)abstract
- Background: The wide range of antibody specificity and affinity results from the differing shapes and chemical compositions of their binding sites. These shapes range from discrete grooves in antibodies elicited by linear oligomers of nucleotides and carbohydrates to shallow depressions or flat surfaces for accommodation of proteins: peptides and large organic compounds. Objectives: To determine the Fab structure of a high-affinity human antitoxin antibody. To explore structural features which enable the antibody to bind to intact tetanus toxoid, peptides derived from the sequence of the natural immunogen and antigenic mimics identified by combinatorial chemistry. To explain why this Fab shows a remarkable tendency to produce crystals consistently diffracting to d spacings of 1.7-1.8 Å. To use this information to engineer a strong tendency to crystallize into the design of other Fabs. Study design: The protein was crystallized in hanging or sitting drops by a microseeding technique in polyethylene glycol (PEG) 8000. Crystals were subjected to X-ray analysis and the three-dimensional structure of the Fab was determined by the molecular replacement method. Interactive computer graphics were employed to fit models to electron density maps, survey the structure in multiple views and discover the crystal packing motif of the protein. Results: Exceptionally large single crystals of this protein have been obtained, one measuring 5 x 3 x 2 mm (l x w x d). The latter was cut into six irregular pieces, each retaining the features of the original in diffracting to high resolution (1.8 Å) with little decay in the X-ray beam. In an individual Fab, the active site is relatively flat and it seems likely that the protein antigen and derivative peptides are tightly held on the outer surface without significant penetration into the interior. There is no free space to accommodate even a dipeptide between V(H) and V(L). One of the unique features of the B7-15A2 Fab is a large aliphatic ridge dominating the center of the active site. The CDR3 of the H chain contributes significantly to this ridge, as well as to adjoining regions projected to be important for the docking of the antigen. Both the ease of crystallization and the favorable diffraction properties are mainly attributable to the tight packing of the protein molecules in the crystal lattice. Discussion: The B7-15A2 active site provides a stable and well defined platform for high affinity docking of proteins, peptides and their mimotopes. The advantages for future developments are suggested by the analysis of the crystal properties. It should be possible to incorporate the features promoting crystallization, close packing and resistance to radiation damage into engineered human antibodies without altering the desired specificities and affinities of their active sites.
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| 2. |
- Furebring, Christina, et al.
(författare)
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Evaluation of novel control elements by construction of eukaryotic expression vectors
- 1997
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Ingår i: Gene. - 0378-1119. ; 188:2, s. 191-198
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Tidskriftsartikel (refereegranskat)abstract
- A novel mammalian eukaryotic expression vector for the production of immunoglobulin heavy chain (IgH) genes has been designed. This expression vector contains the variable heavy chain (VH) promoter, the IgH intron enhancer (μE) and the IgH 3' enhancer (3'E). This construct, designated pTIF-1, was stably transfected into the myeloma cell line J558L. A fivefold increase in the expression level of a rearranged IgH gene was observed when using the pTIF-1 vector containing the 3'E compared to an expression vector lacking this enhancer. Interestingly, this positive effect on the expression level of the 3' enhancer appears to be position independent. The introduction of two recently identified Ig control elements, HS3 and HS4, to the vector cassette did not further elevate the expression level in the cell line tested. The pTIF-1 vector can be used for expression of any antibody specificity, using PCR amplification of the VDJ region of interest. Furthermore, the constant region can easily be exchanged, which further facilitates studies to dissect different effector functions of IgH constant genes.
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| 3. |
- Furebring, Christina
(författare)
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Expression of Ig genes. Regulation of transcription and production of human antibodies
- 1996
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- During B lymphocyte development, the transcriptional activity of the IgH locus is subject to spatial and temporal changes. The 3' enhancer (3'E) has been suggested to play an important role in regulation of immunoglobulin gene expression late in B cell development. We have investigated, using transgenic mice, the role of the 3'E in regulating Ig gene expression. Mice harbouring a rearranged IgH gene potentiated by the VH promoter in combination with the IgH intron enhancer (µE), the 3'E or the µE/3'E pair were generated. The 3'E activity is mainly observed in lymphoid tissues and is mainly restricted to the in vivo activated B cells. The 3'E can potentiate Ig gene expression directly in conjunction with the VH promoter. The expression level of the µE/3'E controlled transgene is fivefold higher as compared to the transgene controlled by the µE alone. The aim of my subsequent studies has been to generate and produce human monoclonal antibodies. We have focused our interest on immortalization of the variable region genes, from hybridoma or from a single antigen-specific B cell, using the powerful PCR technique. The variable region genes from single B cells can be immortalized directly or after a cellular amplification step, involving the EL-4 and CD40 cell culture systems. The variable region genes obtained can thereafter be expressed either as Ab fragments, in prokaryotic host cells, or as the entire Ab, in eukaryotic host cells. To allow efficient expression of intact Ab we have optimized a eukaryotic IgH gene expression vector using different combinations of regulatory elements.
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