Purification of his-tagged proteins using printed monolith adsorption columns.

Bio-separation is a crucial process in biotechnology and biochemical engineering for separating biological macromolecules, and the field has long relied on bead-based and expanded bed chromatography. Printed monolith adsorption (PMA) is a new alternative to which uses a 3D-printed monolithic structure containing self-supporting, ordered flow channels. PMA allows for direct purification of biological molecules from crude cell lysates and cell cultures, and like the other technologies, can functionalized to specifically target a molecule and enable affinity chromatography. Here we have combined PMA technology with an immobilized metal affinity ligand (iminodiacetic acid) to provide selectivity of binding to polyhistidine-tagged proteins during PMA chromatography. Two different PMA structures were created and tested for both static and dynamic protein-binding capacity. At comparative linear flow rates, the dynamic binding capacity of both columns was ≈3 mg/mL, while static capacity was shown to differentiate based on column voidage. We show that a polyhistidine-tagged protein can be directly purified from crude lysate with comparable results to the available commercial providers of IMAC, and with a substantially reduced purification time.

Allosteric inhibition of Staphylococcus aureus MenD by 1,4-dihydroxy naphthoic acid: a feedback inhibition mechanism of the menaquinone biosynthesis pathway.

Menaquinones (MKs) are electron carriers in bacterial respiratory chains. In Staphylococcus aureus (Sau), MKs are essential for aerobic and anaerobic respiration. As MKs are redox-active, their biosynthesis likely requires tight regulation to prevent disruption of cellular redox balance. We recently found that the Mycobacterium tuberculosis MenD, the first committed enzyme of the MK biosynthesis pathway, is allosterically inhibited by the downstream metabolite 1,4-dihydroxy-2-naphthoic acid (DHNA). To understand if this is a conserved mechanism in phylogenetically distant genera that also use MK, we investigated whether the Sau-MenD is allosterically inhibited by DHNA. Our results show that DHNA binds to and inhibits the SEPHCHC synthase activity of Sau-MenD enzymes. We identified residues in the DHNA binding pocket that are important for catalysis (Arg98, Lys283, Lys309) and inhibition (Arg98, Lys283). Furthermore, we showed that exogenous DHNA inhibits the growth of Sau, an effect that can be rescued by supplementing the growth medium with MK-4. Our results demonstrate that, despite a lack of strict conservation of the DHNA binding pocket between Mtb-MenD and Sau-MenD, feedback inhibition by DHNA is a conserved mechanism in Sau-MenD and hence the Sau MK biosynthesis pathway. These findings may have implications for the development of anti-staphylococcal agents targeting MK biosynthesis. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

The structure of His-tagged Geobacillus stearothermophilus purine nucleoside phosphorylase reveals a `spanner in the works'.

The 1.72 Šresolution structure of purine nucleoside phosphorylase from Geobacillus stearothermophilus, a thermostable protein of potential interest for the biocatalytic synthesis of antiviral nucleoside compounds, is reported. The structure of the N-terminally His-tagged enzyme is a hexamer, as is typical of bacterial homologues, with a trimer-of-dimers arrangement. Unexpectedly, several residues of the recombinant tobacco etch virus protease (rTEV) cleavage site from the N-terminal tag are located in the active site of the neighbouring subunit in the dimer. Key to this interaction is a tyrosine residue, which sits where the nucleoside ring of the substrate would normally be located. Tag binding appears to be driven by a combination of enthalpic, entropic and proximity effects, which convey a particularly high affinity in the crystallized form. Attempts to cleave the tag in solution yielded only a small fraction of untagged protein, suggesting that the enzyme predominantly exists in the tag-bound form in solution, preventing rTEV from accessing the cleavage site. However, the tagged protein retained some activity in solution, suggesting that the tag does not completely block the active site, but may act as a competitive inhibitor. This serves as a warning that it is prudent to establish how affinity tags may affect protein structure and function, especially for industrial biocatalytic applications that rely on the efficiency and convenience of one-pot purifications and in cases where tag removal is difficult.

Molecular basis for the recognition of steroidogenic acute regulatory protein by the 14-3-3 protein family.

Steroidogenesis in adrenals and gonads starts from cholesterol transport to mitochondria. This is mediated by the steroidogenic acute regulatory protein (STARD1), containing a mitochondrial import sequence followed by a cholesterol-binding START domain. Although mutations in this protein have been linked to lipoid congenital adrenal hyperplasia (LCAH), the mechanism of steroidogenesis regulation by STARD1 remains debatable. It has been hypothesized to involve a molten-globule structural transition and interaction with 14-3-3 proteins. In this study, we aimed to address the structural basis for the 14-3-3-STARD1 interaction. We show that, while the isolated START domain does not interact with 14-3-3, this interaction is enabled by STARD1 phosphorylation at Ser57, close to the mitochondrial peptide cleavage site. Biochemical analysis of the STARD1 affinity toward 14-3-3 and crystal structures of 14-3-3 complexes with Ser57 and Ser195 phosphopeptides suggest distinct roles of site-specific phosphorylations in recruiting 14-3-3, to modulate STARD1 activity, processing and import to the mitochondria. Phosphorylation at Ser195 creates a unique conditional site that could only bind to 14-3-3 upon partial unfolding of the START domain. Overall, our findings on the interaction between 14-3-3 and STARD1 may have potential clinical implications for patients with LCAH.