Figure 5: Comparison of cytoplasmic versus periplasmic IgG expression. | Nature Communications

Figure 5: Comparison of cytoplasmic versus periplasmic IgG expression.

From: Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria

Figure 5

(a) Antigen-binding activity of mFab/hFc anti-MBP cyclonals derived from the cytoplasm of MB1731 cells (closed squares) or E-clonals derived from the periplasm of parental B strain (open squares). Expression was either from plasmid pET21b (red) or pMAZ360 (dark red); signal from cells carrying empty plasmid was used as negative control (green). Data are expressed as the mean±s.e.m. of biological triplicates. (b) Western blot analysis of soluble (sol) and insoluble (ins) fractions derived from the same cells as in a. HC, heavy chain; LC, light chain; asterisk, degraded HC. Mass spectrometry analysis of the degraded HC revealed that the cleavage site is in the CH1 domain of HC, known to fold inefficiently and be the major limiting factor in the assembly of IgG in eukaryotic cells. This bottleneck seems to carry over to SHuffle cells and is likely due to site-specific cleavage of misassembled HC by cytoplasmic protease(s).

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