Prolyl 4-hydroxylase (proline hydroxylase, EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an ␣ 2 ␤ 2 tetramer, the ␤ subunit of which is identical to protein disulfideisomerase (PDI, EC 5.3.4.1). We report here on cloning of the recently discovered ␣(II) subunit from human sources. The mRNA for the ␣(II) subunit was found to be expressed in a variety of human tissues, and the presence of the corresponding polypeptide and the (␣(II)) 2 Prolyl 4-hydroxylase (proline hydroxylase, EC 1.14.11.2) catalyzes the hydroxylation of proline in -Xaa-Pro-Gly-triplets in collagens and other proteins with collagen-like sequences. The enzyme plays a central role in the synthesis of all collagens, as the 4-hydroxyproline residues formed in the reaction are essential for the folding of the newly synthesized collagen polypeptide chains into triple helical molecules. The vertebrate enzyme is an ␣ 2 ␤ 2 tetramer in which the ␣ subunits contribute to most parts of the two catalytic sites (for reviews, see Refs.

BackgroundDisulfide bonds are the most common structural, post-translational modification found in proteins. Antibodies contain up to 25 disulfide bonds depending on type, with scFv fragments containing two disulfides and Fab fragments containing five or six disulfide bonds. The production of antibody fragments that contain native disulfide bonds can be challenging, especially on a large scale. The protein needs to be targeted to prokaryotic periplasm or the eukaryotic endoplasmic reticulum. These compartments are specialised for disulfide bond formation, but both compartments have limitations.ResultsHere we show that the introduction into the cytoplasm of a catalyst of disulfide bond formation and a catalyst of disulfide bond isomerization allows the efficient formation of natively folded scFv and Fab antibody fragments in the cytoplasm of Escherichia coli with intact reducing pathways. Eleven scFv and eleven Fab fragments were screened and ten of each were obtained in yields of >5 mg/L from deep-well plates. Production of eight of the scFv and all ten of the Fab showed a strong dependence on the addition of the folding factors. Yields of purified scFv of up to 240 mg/L and yields of purified Fab fragments of up to 42 mg/L were obtained. Purified fragments showed circular dichroism spectra consistent with being natively folded and were biologically active.ConclusionsOur results show that the efficient production of soluble, biologically active scFv and Fab antibody fragments in the cytoplasm of E. coli is not only possible, but facile. The required components can be easily transferred between different E. coli strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0419-5) contains supplementary material, which is available to authorized users.

Mesenchymal stem/stromal cells (MSCs) have the capacity to counteract excessive inflammatory responses. MSCs possess a range of immunomodulatory mechanisms, which can be deployed in response to signals in a particular environment and in concert with other immune cells. One immunosuppressive mechanism, not so well-known in MSCs, is mediated via adenosinergic pathway by ectonucleotidases CD73 and CD39. In this study, we demonstrate that adenosine is actively produced from adenosine 5 0 -monophosphate (AMP) by CD73 on MSCs and MSC-derived extracellular vesicles (EVs). Our results indicate that although MSCs express CD39 at low level and it colocalizes with CD73 in bulge areas of membranes, the most efficient adenosine production from adenosine 5 0 -triphosphate (ATP) requires co-operation of MSCs and activated T cells. Highly CD39 expressing activated T cells produce AMP from ATP and MSCs produce adenosine from AMP via CD73 activity. Furthermore, adenosinergic signaling plays a role in suppression of T cell proliferation in vitro. In conclusion, this study shows that adenosinergic signaling is an important immunoregulatory mechanism of MSCs, especially in situations where ATP is present in the extracellular environment, like in tissue injury. An efficient production of immunosuppressive adenosine is dependent on the concerted action of CD39-positive immune cells with CD73-positive cells such as MSCs or their EVs. STEM CELLS 2016;34:781-790 SIGNIFICANCE STATEMENTWe have studied immunomodulatory mechanism not so well known in human mesenchymal stromal cells (MSCs), namely adenosinergic signaling mediated by ectonucleotidases CD73 and CD39. We believe that adenosinergic signaling is particularly important mechanism of MSCs in tissue damage where nucleotides such as ATP are abundantly available in the extracellular environment. Human MSCs and MSC-derived extracellular vesicles, which highly express CD73, efficiently produce adenosine from AMP. However, MSCs and CD39-expressing immune cells (such as activated T cells) co-operate in the production of adenosine from ATP. In addition, MSCs can suppress T cell proliferation in an in vitro assay via adenosinergic signaling, when ATP is added to the assay. This mechanism may have been overlooked in the standard potency assays and also in vivo.