RNA‐dependent chaperone (chaperna) as an engineered pro‐region for the folding of recombinant microbial transglutaminase

Abstract

Transglutaminase (TGase) induces the cross‐linking of proteins by catalyzing an acyl transfer reaction. TGase is a zymogen, activated by the removal of its pro‐region. Because the pro‐region is crucial for folding and inhibition of the TGase activity, the recombinant expression of the mature TGase (mTGase) without the pro‐region, usually results in inactive inclusion bodies or low protein yield. Here, Streptomyces netropsis TGase was fused with Escherichia coli lysyl‐tRNA synthetase (LysRS), as a module with chaperoning activity in an RNA dependent manner (chaperna). The TGase activity from purified fusion protein induced via the removal of LysRS by tev protease in vitro. Moreover, active mTGase was produced in E. coli via an intracellular cleavage system, wherein LysRS‐mTGase was cleaved by the coexpressed tev protease in vivo. The results suggest that LysRS essentially mimics pro‐region, which exerts a dual function—folding of TGase into active conformation and keeping it as dormant state—in an RNA‐dependent manner. Thus, trans‐acting RNAs, prompt the cis‐acting chaperone function of LysRS, while being mechanistically similar to the intramolecular chaperone function of the pro‐region. These results could be implemented and extended for the folding of “difficult‐to‐express” recombinant proteins, by harnessing the chaperna function.

A 33-residue peptide tag increases solubility and stability of Escherichia coli produced single-chain antibody fragments

“…5 and 6d ). We also found no positive evidence for an interaction of the P17 tag with RNA, which could contribute to chaperoning 41 – 44 .…”

“…The solubility-enhancing ability of the P17 tag may be endowed by recruiting other molecules such as chaperones or perhaps tRNAs 41 – 44 to promote protein folding. If so, adding two or three copies of the P17 tag might further elevate the solubility of a test scFv.…”

“…S 1b ), indicating an at most moderate confidence in the predicted α-helix. However, the α-helix might still form in the scFv context or be induced by interaction with bacterial components, notably the abundant tRNA, as in the recently described “chaperna” mechanism where RNA is crucial for chaperoning 41 – 44 . Different from the RNA interaction domain used there, we could not detect a stable association of the P17 tag with RNA (Supplementary Fig.…”

TAR RNA Mediated Folding of a Single-Arginine-Mutant HIV-1 Tat Protein within HeLa Cells Experiencing Intracellular Crowding

“…These properties of RNA can stabilize intermediate folding and interfere with protein aggregation. This suggests that, although our understanding of the roles of various molecular chaperones is still limited, as the important role of virally encoded RNA in establishing proteome linkages is considered, there may be a variety of viral-encoding chaperones that have not yet been characterized [ 77 , 78 ].…”

Intein-mediated intracellular production of active microbial transglutaminase in Corynebacterium glutamicum

“…The pro-peptide is essential for zymogen folding but must be posttranslationally cleaved to activate the zymogen [14]. Therefore, zymogen consisting of the propeptide and mature part of mTGase was normally expressed rst and the protease treatment was subsequently applied to remove the pro-peptide [15][16][17][18][19][20][21][22][23][24][25]. The protease treatment was either conducted in vitro [15-18, 20, 22, 23, 25] or accomplished through co-expression of the protease in vivo [19,21,24].…”