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Background Recombinant protein purification is a crucial step for biochemistry and structural biology fields. Rapid robust purification methods utilize various peptide or protein tags fused to the target protein for affinity purification using corresponding matrices and to enhance solubility. However, affinity/solubility-tags often need to be removed in order to conduct functional and structural studies, adding complexities to purification protocols. Results In this work, the Vibrio cholerae MARTX toxin Cysteine Protease Domain (CPD) was inserted in a ligation-independent cloning (LIC) vector to create a C-terminal 6xHis-tagged inducible autoprocessing enzyme tag, called “the CPD-tag”. The pCPD and alternative pCPD/ccdB cloning vectors allow for easy insertion of DNA and expression of the target protein fused to the CPD-tag, which is removed at the end of the purification step by addition of the inexpensive small molecule inositol hexakisphosphate to induce CPD autoprocessing. This process is demonstrated using a small bacterial membrane localization domain and for high yield purification of the eukaryotic small GTPase KRas. Subsequently, pCPD was tested with 40 proteins or sub-domains selected from a high throughput crystallization pipeline. Conclusion pCPD vectors are easily used LIC compatible vectors for expression of recombinant proteins with a C-terminal CPD/6xHis-tag. Although intended only as a strategy for rapid tag removal, this pilot study revealed the CPD-tag may also increase expression and solubility of some recombinant proteins. Electronic supplementary material The online version of this article (doi:10.1186/s12896-016-0323-4) contains supplementary material, which is available to authorized users.

By screening an EMS mutagenised common bean population, we had previously identified the lpa (low phytic acid) 280-10 line that carries a monogenic recessive mutation conferring a 90% reduction of InsP6 content and, differently from most other lpa mutants, does not display negative pleiotropic effects associated to the mutation. In the present work we mapped the lpa-280-10 mutation on bean chromosome 1, in a region syntenic to soybean (Glycine max) chromosomes 19 and 3, where the lpa mutant CX1834 was previously mapped and correlated to multi-drug resistance protein (MRP) type ATP-binding cassette (ABC) transporter gene orthologous to Arabidopsis AtMRP5/AtABCC5 and maize ZmMRP4 (lpa1). Fine mapping of bean lpa-280-10 and DNA sequencing data showed the occurrence of a single amino acid substitution (E to K) in the orthologous PvMRP-1 gene. We also provide data on a second MRP gene (PvMRP-2) present both in soybean and bean genomes, which, despite the high sequence identity (90%) and a very similar pattern of expression during seed development to PvMRP-1, is not able to complement the lpa mutation, suggesting that it may have a different role in the plant. Transcriptional and metabolic analysis of phytic acid pathway demonstrated the existence of a negative feedback mechanism of InsP6, or its degradation products, acting on genes coding for key enzymes of phytic acid pathway and leading to a 30% reduction of myo-inositol. Our results show that the reduced content of myo-inositol is responsible of the ABA hypersensitive response found in lpa-280-10 seed germination, indicating a key role of this metabolite in the multiple pleiotropic effects often reported for lpa mutants.

We have cloned and characterized four Itpk genes from soybean. All four recombinant Itpk proteins showed canonical Ins(1,3,4)P3 5/6-kinase activity, but a kinetic analysis raised questions about its biological significance. Instead, we provide evidence that one alternative biological role for soybean Itpks is to interconvert the Cl− channel inhibitor, Ins(3,4,5,6)P4, and its metabolic precursor, Ins(1,3,4,5,6)P5, within a substrate cycle. The soybean Itpks also phosphorylated Ins(3,4,6)P3 to Ins(1,3,4,6)P4 which was further phosphorylated to Ins(1,3,4,5,6)P5 by soybean Ipk2. Thus, soybean Itpks may participate in an inositol lipid-independent pathway of InsP6 synthesis.

The ARGRIII gene of Saccharomyces cerevisiae encodes a transcriptional regulator that also has inositol polyphosphate multikinase (ipmk) activity [Saiardi et al. (1999) Curr. Biol. 9, 1323-1326]. To investigate how inositol phosphates regulate gene expression, we disrupted the ARGRIII gene. This mutation impaired nuclear mRNA export, slowed cell growth, increased cellular [InsP(3)] 170-fold and decreased [InsP(6)] 100-fold, indicating reduced phosphorylation of InsP(3) to InsP(6). Levels of diphosphoinositol polyphosphates were decreased much less dramatically than was InsP(6). Low levels of InsP(6), and considerable quantities of Ins(1,3,4,5)P(4), were synthesized by an ipmk-independent route. Transcriptional control by ipmk reflects that it is a pivotal regulator of nuclear mRNA export via inositol phosphate metabolism.