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. 2022 Jan 13;11:e57974. doi: 10.7554/eLife.57974

Figure 2. β-lactamase enzymes from most classes become unstable in the absence of DsbA.

Figure 2.

(A) Protein levels of disulfide-bond-containing Ambler class A and B β-lactamases are drastically reduced when these enzymes are expressed in E. coli MC1000 dsbA; the amount of the control enzyme L2-1 is unaffected. (B) Protein levels of Class D disulfide-bond-containing β-lactamases are unaffected by the absence of DsbA. OXA-4 is detected as two bands at ~28 kDa. For panels (A) and (B) protein levels of StrepII-tagged β-lactamases were assessed using a Strep-Tactin-AP conjugate or a Strep-Tactin-HRP conjugate. A representative blot from three biological experiments, each conducted as a single technical repeat, is shown; molecular weight markers (M) are on the left, DnaK was used as a loading control and solid black lines indicate where the membrane was cut. (C) The hydrolytic activities of the tested Class D β-lactamases and of the Class A enzyme GES-1, which could not be detected by immunoblotting, are significantly reduced in the absence of DsbA. The hydrolytic activities of strains harboring the empty vector or expressing the control enzyme L2-1 show no dependence on DsbA. n = 3 (each conducted in technical duplicate), table shows means ± SD, significance is indicated by * = p < 0.05, ns = non-significant.

Figure 2—source data 1. Original files of the full raw unedited immunoblots used to prepare Figure 2A.
‘Top Panel’ in the file name refers to immunoblots carried out using a Strep-Tactin-AP conjugate, while ‘Bottom Panel’ refers to immunoblots carried out using an anti-DnaK 8E2/2 antibody. ‘Left’ and ‘Right’ in the file names refer to the part of the immunoblot to the left or to the right of the vertical black line shown in the final figure, respectively.
Figure 2—source data 2. Uncropped immunoblots used to prepare Figure 2A.
The figure included in the paper is shown in the center and relevant bands used for each part of the figure are marked with color-coded boxes on the uncropped immunoblots.
Figure 2—source data 3. Original files of the full raw unedited immunoblots used to prepare Figure 2B.
‘Top Panel’ in the file name refers to immunoblots carried out using a Strep-Tactin-AP conjugate or a Strep-Tactin-HRP conjugate, while ‘Bottom Panel’ refers to immunoblots carried out using an anti-DnaK 8E2/2 antibody. ‘Left’, ‘Middle’, and ‘Right’ in the file names refer to the part of the immunoblot to the left, in-between, or to the right of the vertical black lines shown in the final figure, respectively.
Figure 2—source data 4. Uncropped immunoblots used to prepare Figure 2B.
The figure included in the paper is shown in the center and relevant bands used for each part of the figure are marked with color-coded boxes on the uncropped immunoblots.