Investigations on the activation of recombinant microbial pro-transglutaminase: in contrast to proteinase K, dispase removes the histidine-tag

Abstract

In order to produce recombinant microbial transglutaminase (rMTG) which is free of the activating protease, dispase was used to activate the pro-rMTG followed by immobilized metal affinity chromatography (IMAC). As shown by MALDI-MS, the dispase does not only cleave the pro-sequence, but unfortunately also cleaves within the C-terminal histidine-tag. Hence, the active rMTG cannot properly bind to the IMAC material. As an alternative, proteinase K was investigated. This protease was successfully applied for the activation of purified pro-rMTG either as free or immobilized enzyme and the free enzyme was also applicable directly in the crude cell extract of E. coli. Thus, it enables a simple two-step activation/purification procedure resulting in protease-free and almost pure transglutaminase preparations. The protocol has been successfully applied to both, wild-type transglutaminase of Streptomyces mobaraensis as well as to the highly active variant S2P. Proteinase K activates the pro-rMTG without unwanted degradation of the histidine-tag. It turned out to be very important to inhibit proteinase K activity, e.g., by PMSF, prior to protein separation by SDS-PAGE.

Directed Evolution of a Bond‐Forming Enzyme: Ultrahigh‐Throughput Screening of Microbial Transglutaminase Using Yeast Surface Display

“…ESI‐MS analysis of the synthetic construct 2 (Table S2) revealed that dispase cleaved 2 pro not only at its usual site, C ‐terminally from Ser in the SFRAP sequence, but also within the His 6 ‐tag sequence (see Supporting Information, section 2.3 and Figure S11). This observation corroborates previous data . Nonetheless, cells treated with either trypsin or enterokinase showed surface display of the enzyme and biotinylation, indicating that post‐display procession of pro‐mTG ( 4 pro and 5 pro , Table S2) succeeded (Figure b, middle and right panels).…”

“…This observation corroborates previous data. [14] Nonetheless, cells treated with either trypsin or enterokinase showeds urface display of the enzyme and biotinylation, indicating that post-display procession of pro-mTG (4pro and 5pro,T able S2) succeeded (Figure 2b,m iddle and right panels).…”

Transglutamination allows production and characterization of native‐sized ELPylated spider silk proteins from transgenic plants

“…The S2P variant of rMTG from S. mobaraensis was expressed as Pro‐TG in E. coli BL21 in a histidine‐tagged form as described previously (Sommer et al ., ). After cell disruption and activation of Pro‐TG with Proteinase K the transglutaminase was purified by Ni‐IMAC (Sommer et al ., ). By the use of dialysis of active fractions against 50 m m Tris/HCl‐buffer, the TG precipitated.…”

“…Lyophilized monomers were solubilized in 50 m m Tris–HCl, pH 8.0 to a final concentration of 1 mg/mL. An equimolar solution of K‐MaSp1‐100xELP and Q‐MaSp1‐100xELP was treated with 0.5 U of rMTG (Sommer et al ., ) per μL protein solution at 11 °C with gentle shaking. The resulting cross‐linked ELPylated proteins were purified by cITC and desalted via gel filtration (Sephadex G‐50; Sigma‐Aldrich).…”

Role of polyamines, their analogs and transglutaminases in biological and clinical perspectives

“…Applications of TGases in material science require optimized enzymes, for instance with higher thermostability, as addressed by Büttner et al (2011) in this issue. These enzymes can be overproduced and purified on a mass scale as described by Sommer et al (2011).…”