describe the necessary materials needed for fast and efficient protein
expression of the materials required for kinetic analysis of the
binding of the different antibody format to the KLK7 target protein by biolayer interferometry (BLI).
2.1 Expression
Vectors
All cloning steps were performed using standard restriction enzyme
digestion with subsequent ligation. Alternatively, the sequenceand ligation-independent cloning (SLIC) technique according to
Li et al. [17] was used to generate the expression clones. DNA
preparation for transient gene expression was done using the PureYield Plasmid Midiprep kit (Promega) or the endotoxin-free Plasmid DNA Gigaprep kit (Qiagen). The construction of the cloning
vectors is described in detail in J€ ager et al. [16].
1. pCSE2.6-hIgG1-Fc-LUP37-A10 (Expression vector for the
synthesis of the antibody LUP37-A10 in the scFv-Fc format).
2. pCSE2.6-hIgG1-Fc-LUP37-B10 (Expression vector for the synthesis of the antibody LUP37-B10 in the scFv-Fc format).
3. pCSE2.6-hIgG1-Fc-LUP37-C11 (Expression vector for the synthesis of the antibody LUP37-C11 in the scFv-Fc format).
4. pCSE2.6-hIgG1-Fc-LUP37-D11 (Expression vector for the
synthesis of the antibody LUP37-D11 in the scFv-Fc format).
5. pCSL3l (Expression vector for the synthesis of antibody light
chains. VL domains were cloned into this vector for production
of IgG and Fab antibodies).
6. pCSEH1c (Expression vector for the synthesis of antibody
heavy chains. VH domains were cloned into this vector for
production of IgG antibodies).
7. pCSE2.5-Fab-h-HIS-XP (Expression vector for the synthesis of
antibody heavy chains lacking the domains CH2 and CH3. VH
domains were cloned into this vector for the production of
antibodies in the Fab format).
8. pUC57-His8-proKLK-3C-hKLK7 (Cloning vector obtained
from GenScript carrying the synthetic codon optimized DNA
sequence His8-proKLK-3C-hKLK7).
9. pOpIE2- His8-proKLK-3C-hKLK7 (The sE2-dHVR1
sequence of pOpIE2-sE2-dHVR1 was exchanged with the
insert His8-proKLK-3C-hKLK7 to get a Hi5 expression vector
for human proKLK7).
10. pTTo/eGFPq (Control vector for the determination of transfection efficiency in HEK293-6E cells via flow cytometry).
11. pOpiE2-eGFP-HA (Control vector for the determination of
transfection efficiency in Hi5 cells via flow cytometry).
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Jens Ko ¨ nig et al.
expression of the materials required for kinetic analysis of the
binding of the different antibody format to the KLK7 target protein by biolayer interferometry (BLI).
2.1 Expression
Vectors
All cloning steps were performed using standard restriction enzyme
digestion with subsequent ligation. Alternatively, the sequenceand ligation-independent cloning (SLIC) technique according to
Li et al. [17] was used to generate the expression clones. DNA
preparation for transient gene expression was done using the PureYield Plasmid Midiprep kit (Promega) or the endotoxin-free Plasmid DNA Gigaprep kit (Qiagen). The construction of the cloning
vectors is described in detail in J€ ager et al. [16].
1. pCSE2.6-hIgG1-Fc-LUP37-A10 (Expression vector for the
synthesis of the antibody LUP37-A10 in the scFv-Fc format).
2. pCSE2.6-hIgG1-Fc-LUP37-B10 (Expression vector for the synthesis of the antibody LUP37-B10 in the scFv-Fc format).
3. pCSE2.6-hIgG1-Fc-LUP37-C11 (Expression vector for the synthesis of the antibody LUP37-C11 in the scFv-Fc format).
4. pCSE2.6-hIgG1-Fc-LUP37-D11 (Expression vector for the
synthesis of the antibody LUP37-D11 in the scFv-Fc format).
5. pCSL3l (Expression vector for the synthesis of antibody light
chains. VL domains were cloned into this vector for production
of IgG and Fab antibodies).
6. pCSEH1c (Expression vector for the synthesis of antibody
heavy chains. VH domains were cloned into this vector for
production of IgG antibodies).
7. pCSE2.5-Fab-h-HIS-XP (Expression vector for the synthesis of
antibody heavy chains lacking the domains CH2 and CH3. VH
domains were cloned into this vector for the production of
antibodies in the Fab format).
8. pUC57-His8-proKLK-3C-hKLK7 (Cloning vector obtained
from GenScript carrying the synthetic codon optimized DNA
sequence His8-proKLK-3C-hKLK7).
9. pOpIE2- His8-proKLK-3C-hKLK7 (The sE2-dHVR1
sequence of pOpIE2-sE2-dHVR1 was exchanged with the
insert His8-proKLK-3C-hKLK7 to get a Hi5 expression vector
for human proKLK7).
10. pTTo/eGFPq (Control vector for the determination of transfection efficiency in HEK293-6E cells via flow cytometry).
11. pOpiE2-eGFP-HA (Control vector for the determination of
transfection efficiency in Hi5 cells via flow cytometry).
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Jens Ko ¨ nig et al.