Bernhard Auer

Background: Escherichia colias a frequently utilized host organism for recombinant protein production offers different cellular locations with distinct qualities. The periplasmic space is often favored for the production of complex proteins due to enhanced disulfide bond formation, increased target product stability and simplified downstream processing. To direct proteins to the periplasmic space rather small proteinaceus tags that can be used for affinity purification would be advantageous.Results: We discovered that domain D of theStaphylococcus aureusprotein A was sufficient for the secretion of various target proteins into the periplasmic space ofE. coli. Our experiments indicated the Sec pathway as the mode of secretion, although N-terminal processing was not observed. Furthermore, the solubility of recombinant fusion proteins was improved for proteins prone to aggregation.The tag allowed a straightforward affinity purification of recombinant fusion protein via an IgG column, which was exemplified for the target protein human superoxide dismutase 1 (SOD).Conclusions: In this work we present a new secretion tag that combines several advantages for the production of recombinant proteins inE. coli. Domain D ofS. aureusprotein A protects the protein of interest against N-terminal degradation, increases target protein solubility and enables a straight-forward purification of the recombinant protein using of IgG columns.

Background: Many protocols for recombinant production of peptides and proteins include secretion into the periplasmic space of Escherichia coli, as they may not properly fold in the cytoplasm. If a signal peptide is not sufficient for translocation, a larger secretion moiety can instead be fused to the gene of interest. However, due to the covalent linkage of the proteins, a protease recognition site needs to be introduced in between, altering the N-terminus of the product. In the current study, we combined the ubiquitin fusion technology, which allows production of authentic peptides and proteins, with secretion by the perpiplasmic protease inhibitor ecotin. Results: Different fusion constructs, composed of ecotin, mouse ubiquitin b and a model peptide, were expressed in E. coli BL21(DE3). The fusion proteins were translocated into the periplasmic space and the ecotin signal peptide was cleaved off. Under the control of the lacUV5 promoter at 24°C we obtained 18 mg periplasmic recombinant protein per gram dry cell weight. However, vigorous expression with the T7 promoter caused outer membrane permeabilization and leakage of the fusion protein into the culture medium. Target peptides were released from hybrid proteins by the deubiquitinating enzyme ubiquitin c-terminal hydrolase-L3 in vitro. MALDI TOF-TOF mass spectroscopy confirmed accurate cleavage. Conclusion: This newly described method represents a useful technique for the production of authentic soluble peptides in the periplasm of E. coli. In addition, larger proteins might also be produced with the current system by the use of ubiquitin specific proteases, which can cleave off larger C-terminal extensions.

Nonenzymatic ADP‐ribosylation of mitochondrial proteins is thought to play a role in the regulation of Ca²⁺ efflux from mitochondria. It has been shown that intramitochondrial ADP‐ribose is generated by a specific NAD⁺ glycohydrolase, which catalizes hydrolysis of NAD⁺ to ADP‐ribose and nicotinamide. We purified this enzyme from bovine liver mitochondrial membranes. The final preparation had a 1660‐fold purified enzyme activity and contained a main protein band with an apparent molar mass of 32,000 in a SDS‐polyacrylamide gel. The identity of this protein band with NAD⁺‐glycohydrolase was verified by renaturation of its enzymatic activity. Partial amino acid sequence information was obtained from two enzyme fragments after proteolytic cleavage of the protein band in the SDS‐polyacrylamide gel. Searches in protein databases revealed that an arginine ADP‐ribosyl hydrolase harbours two stretches of amino acids that are highly similar to the partial NAD⁺‐glycohydrolase sequences.