Table 1.
Primers used to construct the N-terminal tags.
Table 2.
Fusion sites used in the MoClo Toolkit (Addgene, kit # 1000000044) and the numbers attributed for their identification on the pET28g cloning system.
Fig 1.
Schematic representation of pET28g assembly.
(A) The Golden Gate cassette was amplified by PCR and digested with NcoI and XhoI enzymes, exposing the sticky ends that match those exposed in the (B) pET28a digested with the same pair of restriction enzymes. (C) The pET28g was obtained through ligation of the digested DNA and contains a lacZα reporter gene flanked by BsaI recognition sites that expose the sticky ends of fusion sites 1 (FS1) and 7 (FS7) (Table 2) used for level 1 assembly.
Fig 2.
Strategy to clone level 0 modules using the MoClo Toolkit (Addgene, kit #1000000044) universal level 0 acceptor plasmid (pAGM9121).
(A) Schematic representation of the level 0 acceptor plasmid and the cleavage site of the BpiI enzyme; (B) Schematic representations of different approaches to prepare the FOI for cloning: (B1) Synthesize the DNA sequence of interest; (B2) Design primers to amplify the DNA of interest; (B3) If DNA size < 100 bp, design oligonucleotides complementary for the DNA of interest region, flanked by uncomplemented fusion sites; (C) Schematic representation of the final plasmid obtained upon ligation of the FOI (B) to the universal acceptor plasmid (A) using BpiI for Golden Gate assembly. The BsaI sites reconstituted in the final plasmid are italicized.
Fig 3.
Strategy to clone level 0 modules using a MoClo Toolkit (Addgene, kit #1000000044) plasmid with the predefined fusion sites (pAGM1299 with fusion sites 3 and 4 was used as an example).
(A) Schematic representation of the level 0 acceptor and the cleavage site of BpiI and BsaI enzymes; (B) Schematic representations of different approaches to preparing the FOI for cloning: (B1) Synthesize the DNA sequence of interest; (B2) Design primers to amplify the DNA of interest; (B3) If DNA size < 100 bp, design primers to amplify the DNA of interest,design oligonucleotides complementary for the DNA of interest region, flanked by uncomplemented fusion sites; (C) Schematic representation of the final vector obtained upon ligation of the FOI (B) to the universal acceptor vector (A) using BpiI for Golden Gate assembly.
Fig 4.
Detection of ACE2 expressed in E. coli BL21 (DE3) using different constructs.
Protein expression as detected in total soluble protein extracts by western blot with anti-ACE2 antibody (A and B) and Coomassie Brilliant Blue staining (C and D). A and C, soluble protein extracts of bacteria transformed with pET28a plasmid expressing ACE2 with a 6x-His tag at the N-terminus; B and D, soluble protein extracts of bacteria transformed with pET28g plasmids expressing ACE2 with an 8 × His-Gb1 tag (Gb1) or an MBP tag (MBP) at the N-terminus. NI – Soluble protein extract from bacteria before protein expression induction; I4 – Soluble protein extract from bacteria four hours after protein expression induction; M – Molecular weight marker (MB09002, NZYTech).
Fig 5.
Purification of ACE2 expressed in E. coli BL21 (DE3) using a pET28g plasmid with an 8
× His-Gb1 tag. A) SDS-PAGE analysis of fractions from the reverse HisTrap purification step, performed after TEV protease cleavage to remove the 8 × His-GB1 tag and His-tagged TEV protease. The flowthrough (FT) contains the cleaved (untagged) ACE2, while the elution fraction contains the His-tagged TEV protease and the cleaved His-tag. Washes 1 and 2 (W1, W2) with PBS pH 7.4 ensured complete recovery of untagged ACE2. The figure also shows fractions (a) and (b) collected from size-exclusion chromatography. (B) Size-exclusion chromatography profile of untagged ACE2 purified using a HiLoad 16/600 Superdex™ 200 pg column, where peak (b) corresponds to the untagged ACE2 protein. M – molecular weight marker (MB09002, NZYTech).
Fig 6.
Assessment of the secondary structure content and enzymatic activity of recombinantly expressed ACE2 in E. coli.
(A) Far-UV CD spectra of ACE2 with a characteristic α-helical profile, indicating a predominantly helical secondary structure. Measurements were performed at a protein concentration of 0.2 mg/mL in PBS, pH 7.4, using a 0.1 cm path length cuvette at 25°C. Baseline correction was applied using the buffer CD spectrum. (B) Rates of Mca-APK(Dnp) substrate cleavage by recombinant ACE2. The rates are given in fluorescence arbitrary units (a.u.) because the unquenched Mca substrate was not available to prepare a calibration curve. For comparison, the rates observed for the control reactions, in the absence of substrate or protein, were plotted. Differences between the rate determined for ACE2 and the controls are statistically significant.