Self-processing of a barley subtilase expressed in E. coli

Abstract

The barley protease BAJ93208 belongs to the subtilase family of serine proteases. We have expressed BAJ93208 in the cytoplasm of the Escherichiacoli strain SHuffle C3030 using a rhamnose-inducible promoter. The expression construct included a (His)6-tag at the N-terminus and a strep-tag at the C-terminus. Western blot analysis revealed that the protein was processed at the N- and C-terminus. To exclude that this processing was due to contaminating E. coli proteases, a mutated BAJ93208 protease was constructed. This inactive mutant was not processed, demonstrating that the processing was an autocatalytic process. To define the exact cleavage sites mass spectrometry was used which detected four differently processed versions of the protease. At the N-terminus, the self-processing removed the internal inhibitor and an additional 19 amino acids. At the C-terminus there was a cleavage site after Ala(765) which also removed the strep-tag. This explained the inability to detect the purified (His)6-BAJ93208-strep protease with an anti-strep-tag antibody. Finally, an additional alanine was removed either at the N-terminus (Ala(119)) or at the C-terminus (Ala(764)).

Keywords: Proprotein; Subtilase; Thionin.

Fig. 1

(A) Purification of (His)₆-BAJ93208-strep on Strep-tactin Sepharose column. SDS–PAGE showing (M) protein ladder, (UN) uninduced E. coli cells, (IN) induced E. coli cells, (F1–F4) eluted fractions from strep-tactin column. (B) SDS–PAGE and Multiplex Western blot detection of N-terminal (His)₆-tag and C-terminal strep-tag of (His)₆-BAJ93208-Strep. M: marker, IN: IPTG induced E. coli cells expressing (His)₆-BAJ93208-strep, F3: strep-tag purified (His)₆-BAJ93208-strep sample. During the purification process the protease is partially processed at the N-terminus. Star indicates (His)₆-BAJ93208-Strep and triangle shows BAJ93208-Strep without inhibitor.

Fig. 2

(A) Elution chromatogram of the anion exchange purification of pooled fractions F1–F4 on a Mono Q column. (B) SDS–PAGE analysis of fractions from anion exchange chromatography of (His)₆-BAJ93208-strep. One single band can be found in protease active fractions A6-A11. Star indicates (His)₆-BAJ93208-Strep and triangle shows BAJ93208-Strep without inhibitor.

Fig. 3

(A) LC–ESI–MS analysis revealed four different truncated versions of the purified protease. (B) Sketch of the recombinant BAJ93208 protease with calculated average masses [M+H]⁺¹ for the identified digestion products (all 10 cysteines in oxidized form). Two cleavages occur at the N-terminus removing the inhibitor and 19 additional amino acids. Two cleavages occur at the C-terminus releasing the strep-tag and to some extent an additional alanine residue. Domain structures predicted according to the architecture prediction program SMART. HIS: (His)₆-tag, I9: inhibitor, S8: peptidase domain, PA: protease associated domain, FN3: fibronectin III domain, strep: strep-tag. (C) Amino acid sequence of the double-tagged protease precursor. Tags are underlined. The inhibitor is shown in bold and the additional 19 amino acids which are cleaved off at the N-terminus after removal of the inhibitor are italic. The amino acids constituting the active site are shown in bold and shaded. ▾ indicates cleavage sites. indicates possible cleavage site which would remove one alanine.

Fig. 4

(A) SDS–PAGE and Multiplex Western blot detecting the N-terminal (His)₆-tag and the C-terminal strep-tag. IN: IPTG induced E. coli cells expressing the mutated (His)₆-BAJ93208-strep (indicated by a star), F3: strep-tag purified mutated (His)₆-BAJ93208-strep sample. The blot shows that the mutated protease is not processed during the purification process. (B) Activity analysis of BJ93208-strep and mutated (His)₆-BJ93208-strep proteases using the fluorogenic peptide as substrate. The mutated version of the protease shows no activity.