Effects of unpaired cysteines on yield, solubility and activity of different recombinant antibody constructs expressed in E. coli.
New E. coli vectors based on the pOPE/pSTE vector system [Gene 128 (1993) 97] were constructed to express a single-chain Fv antibody fragment (scFv), a scFv-streptavidin fusion protein and two disulfide bond-stabilized Fv antibody fragments (dsFvs) utilizing different side chain positions for disulfide stabilization. All of these constructs encoded fusion proteins carrying five C-terminal histidine residues preceded by an unpaired cysteine. The influence of this cysteine, which was originally introduced to allow the chemical modification of the fusion proteins, was assessed by exchanging the two amino acids CysIle in front of the carboxy terminal His-tag to SerHis in all constructs. Yield and antigen-binding activity of the antibody constructs were compared after standard lab-scale periplasmic expression in Escherichia coli. The removal of the unpaired cysteine resulted in a significant increase in antigen-binding activity of the crude periplasmic extracts. Further, a three-five fold increase of yield and a significantly improved purity were observed after immobilized metal affinity chromatography (IMAC) with all four constructs.
Schmiedl
A
A
Universität Heidelberg, Institut für Molekulare Genetik, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany.
Breitling
F
F
Winter
C H
CH
Queitsch
I
I
Dübel
S
S
D000595:Amino Acid Sequence; D000917:Antibody Formation; D000937:Antigen-Antibody Reactions; D003545:Cysteine / Q000276:immunology*; D004926:Escherichia coli; D015870:Gene Expression; D005822:Genetic Vectors; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000276:immunology*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012995:Solubility
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; D003545:Cysteine