Engineered pH‐inducible intein Mtu ΔI‐CM variants with markedly reduced premature cleavage activity

Abstract

Inteins have been widely exploited for the purification of tagless proteins. Among them, pH‐inducible C‐terminal‐cleavage inteins enable the preparation of proteins and peptides with an authentic N‐terminus. However, a severe premature cleavage around neutral pH has limited the application of these inteins, especially when used in recombinant hosts such as Escherichia coli. By targeting the microenvironment of the two key histidine residues H429 and H439, we engineered Mtu ΔI‐CM intein to markedly reduce its premature cleavage. Kinetic analyses suggested that although the variants retained the pH dependence, they indeed cleaved slower, especially at pH 7.6. These variants resulted in higher yields for two model polypeptides than the original Mtu ΔI‐CM intein, when used in conjunction with a cleavable self‐assembling tag. This work suggests that more controllable pH‐inducible inteins can be obtained by manipulating the residues in the self‐cleavage sites and provide better performance for tag‐based protein preparation strategies.

Spy chemistry‐enabled protein directional immobilization and protein purification

“…The protein purification scheme was investigated using RFP, and the variable domain of the heavy chain antibody from llama targeting the dengue virus Type 2 NS1 protein (Fatima et al, 2014), as the model proteins. The 18.5 kDa intein variant Mtu ΔI-CM H73V/T430S, which was previously evolved to reduce the self-cleavage activity in vivo (Lin et al, 2019), was used to generate POIs with authentic N-termini.…”

“…This scheme was then further explored as a protein purification strategy using RFP and a variable domain of a heavy chain antibody (VHH) as model proteins. As shown in Figure 1c, SpyTag is chemically synthesized directly on resin, whereas the POI is fused with SpyCatcher and a self-cleaving Mtu intein (Lin et al, 2019). The tri-fusion protein (hereinafter referred to as SpyCatcher-Mtu-POI) is first captured by the SpyTag-modified resin, followed by the pH-induced intein self-cleavage to release the POI with an authentic N-terminus.…”

Customizable Click Biochemistry Strategy for the Design and Preparation of Glucagon-like Peptide-1 Conjugates and Coagonists

“…Furthermore, the pH-inducible C-terminal cleavage intein, Mtu ΔI-CM, used in the icSAT scheme enables the generation of GLP-1 or GIP with an authentic N-terminus, which is crucial for their agonist activity. , The premature cleavage of this intein is predominantly modulated by the N-terminal residues of the fusion peptide or protein partner adjacent to the intein, particularly histidine, cysteine, serine, or threonine which can donate protons at intercellular neutral pH. , Thus, further engineering of Mtu ΔI-CM, or the use of an alternative C-terminal cleavage intein capable of self-cleavage upon the addition of thiol agents ( e.g., Sce VMA), can be considered . Additionally, the utilization of the SpyCatcher-SpyTag complex can also be extended to other protein-peptide reaction pairs, such as SnoopCatcher-SnoopTag and SdyCatcher-SdyTag …”

“…For the intein, we selected the variant Mtu ΔI-CM (m2) (H73 V/T430S) to minimize premature cleavage of Mtu ΔI-CM-GLP-1. 27 For the scaffold proteins, we used the low-immunogenic SpyCatcher(ΔN) units flanking with ELP 15 domains consisting of 15 repeating Val-Pro-Gly-Xaa-Gly units, where Xaa is Val, Glu, or Val at a ratio of 2:1:2. 21,24,28 The strategy for preparing Di-GLP-1 or Tri-GLP-1 is shown in Figure 1B.…”

“…The icSAT scheme was modified from previous study. 27 Briefly, icSATtagged Di-GLP-1, Tri-GLP-1, or GLP-1/GIP was mixed with an equal volume of high salt buffer B2 (20 mM Tris, 1 mM EDTA, 1.4 M Na 2 SO 4 , pH 8.0), respectively, and incubated overnight at 4 °C. The resulting mixture was subsequently centrifuged at 15,000 g for 20 min at 4 °C.…”