Preparation of cell-permeable Cre recombinase by expressed protein ligation

Background

Protein transduction is safer than viral vector-mediated transduction for the delivery of a therapeutic protein into a cell. Fusion proteins with an arginine-rich cell-penetrating peptide have been produced in E. coli, but the low solubility of the fusion protein expressed in E. coli impedes the large-scale production of fusion proteins from E. coli.

Results

Expressed protein ligation is a semisynthetic method to ligate a bacterially expressed protein with a chemically synthesized peptide. In this study, we developed expressed protein ligation-based techniques to conjugate synthetic polyarginine peptides to Cre recombinase. The conjugation efficiency of this technique was higher than 80%. Using this method, we prepared semisynthetic Cre with poly-L-arginine (ssCre-R9), poly-D-arginine (ssCre-dR9) and biotin (ssCre-dR9-biotin). We found that ssCre-R9 was delivered to the cell to a comparable level or more efficiently compared with Cre-R11 and TAT-Cre expressed as recombinant fusion proteins in E. coli. We also found that the poly-D-arginine cell-penetrating peptide was more effective than the poly-L-arginine cell-penetrating peptide for the delivery of Cre into cell. We visualized the cell transduced with ssCre-dR9-biotin using avidin-FITC.

Conclusions

Collectively, the results demonstrate that expressed protein ligation is an excellent technique for the production of cell-permeable Cre recombinase with polyarginine cell-penetrating peptides. In addition, this approach will extend the use of cell-permeable proteins to more sophisticated applications, such as cell imaging.

Electronic supplementary material

The online version of this article (doi:10.1186/s12896-015-0126-z) contains supplementary material, which is available to authorized users.     

Sensing of mycobacterial arabinogalactan by galectin‐9 exacerbates mycobacterial infection

Mycobacterial arabinogalactan (AG) is an essential cell wall component of mycobacteria and a frequent structural and bio‐synthetical target for anti‐tuberculosis (TB) drug development. Here, we report that mycobacterial AG is recognized by galectin‐9 and exacerbates mycobacterial infection. Administration of AG‐specific aptamers inhibits cellular infiltration caused by Mycobacterium tuberculosis (Mtb) or Mycobacterium bovis BCG, and moderately increases survival of Mtb‐infected mice or Mycobacterium marinum‐infected zebrafish. AG interacts with carbohydrate recognition domain (CRD) 2 of galectin‐9 with high affinity, and galectin‐9 associates with transforming growth factor β‐activated kinase 1 (TAK1) via CRD2 to trigger subsequent activation of extracellular signal‐regulated kinase (ERK) as well as induction of the expression of matrix metalloproteinases (MMPs). Moreover, deletion of galectin‐9 or inhibition of MMPs blocks AG‐induced pathological impairments in the lung, and the AG‐galectin‐9 axis aggravates the process of Mtb infection in mice. These results demonstrate that AG is an important virulence factor of mycobacteria and galectin‐9 is a novel receptor for Mtb and other mycobacteria, paving the way for the development of novel effective TB immune modulators.     

The Wnt receptor Ryk controls specification of GABAergic neurons versus oligodendrocytes during telencephalon development

GABAergic neurons and oligodendrocytes originate from progenitors within the ventral telencephalon. However, the molecular mechanisms that control neuron-glial cell-fate segregation, especially how extrinsic factors regulate cell-fate changes, are poorly understood. We have discovered that the Wnt receptor Ryk promotes GABAergic neuron production while repressing oligodendrocyte formation in the ventral telencephalon. We demonstrate that Ryk controls the cell-fate switch by negatively regulating expression of the intrinsic oligodendrogenic factor Olig2 while inducing expression of the interneuron fate determinant Dlx2. In addition, we demonstrate that Ryk is required for GABAergic neuron induction and oligodendrogenesis inhibition caused by Wnt3a stimulation. Furthermore, we showed that the cleaved intracellular domain of Ryk is sufficient to regulate the cell-fate switch by regulating the expression of intrinsic cell-fate determinants. These results identify Ryk as a multi-functional receptor that is able to transduce extrinsic cues into progenitor cells, promote GABAergic neuron formation, and inhibit oligodendrogenesis during ventral embryonic brain development.     

Interception of teicoplanin oxidation intermediates yields new antimicrobial scaffolds

In the search for new efficacious antibiotics, biosynthetic engineering offers attractive opportunities to introduce minor alterations to antibiotic structures that may overcome resistance. Dbv29, a flavin-containing oxidase, catalyzes the four-electron oxidation of a vancomycin-like glycopeptide to yield A40926. Structural and biochemical examination of Dbv29 now provides insights into residues that govern flavinylation and activity, protein conformation and reaction mechanism. In particular, the serendipitous discovery of a reaction intermediate in the crystal structure led us to identify an unexpected opportunity to intercept the normal enzyme mechanism at two different points to create new teicoplanin analogs. Using this method, we synthesized families of antibiotic analogs with amidated and aminated lipid chains, some of which showed marked potency and efficacy against multidrug resistant pathogens. This method offers a new strategy for the development of chemical diversity to combat antibacterial resistance.     

Large-scale N-glycoproteome map of rat brain tissue: simultaneous characterization of insoluble and soluble protein fractions

The large-scale N-glycosylation analysis is critical for biomedical research, since a variety of diseases are found to be associated with glycoproteins. By a combination of glycoprotein analysis in insoluble protein fraction solubilized with 1% v/v 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM BF(4)) and those in soluble fraction, a total number of 462 non-redundant N-glycoprotein groups, including 316 transmembrane glycoproteins, were successfully identified. Correspondingly, 849 unique N-glycosites were confidently recognized. The data set could provide a support for the further in-depth research of brain N-glycosylation, such as for the discovery of candidate drug targets and biomarkers.     

利用市政污水培养Chlorella vulgaris生产生物柴油

为了考察利用南昌市政污水规模化培养富油微藻生产生物柴油,同时达到净化污水的目的,取南昌市青山湖污水处理厂未经任何处理的市政污水作为普通小球藻(Chlorella vulgaris)生长的培养液。监测了C.vulgaris在市政污水中连续培养10 d的特定生长率、生物质产量以及与之相关的市政污水中氨氮(NH4+-N)、总磷(TP)、化学需氧量(COD)、总悬浮固体(TSS)和挥发性悬浮固体(VSS)的清除情况。实验表明:营养物质的水平显著地影响了C.vulgaris的生长。C.vulgaris的生长率在培养8 d后达到最大,OD680为2.856,总的生物质产量日均最大积累速率为0.01 g/L,油脂含量为干质量的18%,油脂的平均日产量为0.001 g/L。培养10 d内NH4+-N、TP和COD的去除率分别为50.0%、32.1%和26.0%,TSS和VSS的日平均去除速率分别为0.01 g/L和0.006 1 g/L。     

Polarity changes in the transmembrane domain core of HIV-1 Vpu inhibits its anti-tetherin activity

Tetherin (BST-2/CD317) is an interferon-inducible antiviral protein that restricts the release of enveloped viruses from infected cells. The HIV-1 accessory protein Vpu can efficiently antagonize this restriction. In this study, we analyzed mutations of the transmembrane (TM) domain of Vpu, including deletions and substitutions, to delineate amino acids important for HIV-1 viral particle release and in interactions with tetherin. The mutants had similar subcellular localization patterns with that of wild-type Vpu and were functional with respect to CD4 downregulation. We showed that the hydrophobic binding surface for tetherin lies in the core of the Vpu TM domain. Three consecutive hydrophobic isoleucine residues in the middle region of the Vpu TM domain, I15, I16 and I17, were important for stabilizing the tetherin binding interface and determining its sensitivity to tetherin. Changing the polarity of the amino acids at these positions resulted in severe impairment of Vpu-induced tetherin targeting and antagonism. Taken together, these data reveal a model of specific hydrophobic interactions between Vpu and tetherin, which can be potentially targeted in the development of novel anti-HIV-1 drugs.