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.     

Solution structure of family 21 carbohydrate-binding module from Rhizopus oryzae glucoamylase

Evolutionarily conserved SR proteins (serine/arginine-rich proteins) are important factors for alternative splicing and their activity is modulated by SRPKs (SR protein-specific kinases). We previously identified Dsk1p (dis1-suppressing protein kinase) as the orthologue of human SRPK1 in fission yeast. In addition to its similarity of gene structure to higher eukaryotes, fission yeast Schizosaccharomyces pombe is a unicellular eukaryotic organism in which alternative splicing takes place. In the present study, we have revealed for the first time that SR proteins, Srp1p and Srp2p, are the in vivo substrates of Dsk1p in S. pombe. Moreover, the cellular localization of the SR proteins and Prp2p splicing factor is dependent on dsk1(+): Dsk1p is required for the efficient nuclear localization of Srp2p and Prp2p, while it promotes the cytoplasmic distribution of Srp1p, thereby differentially influencing the destinations of these proteins in the cell. The present study offers the first biochemical and genetic evidence for the in vivo targets of the SRPK1 orthologue, Dsk1p, in S. pombe and the significant correlation between Dsk1p-mediated phosphorylation and the cellular localization of the SR proteins, providing information about the physiological functions of Dsk1p. Furthermore, the results demonstrate that the regulatory function of SRPKs in the nuclear targeting of SR proteins is conserved from fission yeast to human, indicating a general mechanism of reversible phosphorylation to control the activities of SR proteins in RNA metabolism through cellular partitioning.     

四株糖单孢菌分离株的分类学研究

从广西地区的土样中,分离到4株细胞壁Ⅳ型,糖型A, 无枝菌酸的假诺卡氏菌科的放线菌菌株,编号分别为191、221、202、和212。根据4株 菌的形态学特征和细胞化学特征,将其归入糖单孢菌属。与该属5个已知种的7个代表株进行 的rDNA的BamHI酶切片段长度类型分析(Ribotyping)的结果表明：191为青绿色糖单孢菌(S.viridis),202为青灰色糖单孢菌(S.caesia),221和212为相同的与青绿色糖单孢菌(S .viridis)的亲缘关系最近的种,但不同于已知的任何一个种。     

棘冠海星暴发及其对珊瑚礁的生态影响研究进展

棘冠海星的反复暴发是导致印度—太平洋区域珊瑚礁生态系统退化的最主要原因之一。然而,我国对棘冠海星的研究非常有限。本文综述了国内外关于棘冠海星及其暴发的生态影响和应对策略的研究进展,得出以下主要结论：1)雌性棘冠海星个体每年产卵数量高达50万—2亿个,环境因素变化只要导致幼虫和幼体存活率的轻微提高,成体就将得到大量补充;2)棘冠海星暴发的阈值为1000—1500个/km²,暴发周期为10—27 a,每次暴发持续1—10 a,最终可能以“种群集体感染疾病而崩溃”结束;3)棘冠海星暴发对印度洋及太平洋东部和北部珊瑚礁的破坏性非常小,却直接导致太平洋的西部和南部珊瑚礁90%以上的珊瑚死亡,并通过改变珊瑚群落组成、减少珊瑚和鱼类多样性而对珊瑚礁产生间接影响;4)关于棘冠海星暴发原因的假说中“陆地营养物质输入假说”和“捕食者过度捕捞假说”得到了最普遍的认可,但都不能解释所有的暴发事件;5)应对棘冠海星暴发的主要策略有改善水质、设立保护区、投放天敌和人工清理等,其中人工清理是最直接有效的策略,但迄今并没有发现可长期抑制棘冠海星暴发的方法。因此,急需加强对棘冠海星的深入研究,探查...     

Fine mapping of a distal chromosome 4 QTL affecting growth and muscle mass in a chicken advanced intercross line

In our previous research, QTL analysis in an F₂ cross between the inbred New Hampshire (NHI) and White Leghorn (WL77) lines revealed a growth QTL in the distal part of chromosome 4. To physically reduce the chromosomal interval and the number of potential candidate genes, we performed fine mapping using individuals of generations F₁₀, F₁₁ and F₁₂ in an advanced intercross line that had been established from the initial F₂ mapping population. Using nine single nucleotide polymorphism (SNP) markers within the QTL region for an association analysis with several growth traits from hatch to 20 weeks and body composition traits at 20 weeks, we could reduce the confidence interval from 26.9 to 3.4 Mb. Within the fine mapped region, markers rs14490774, rs314961352 and rs318175270 were in full linkage disequilibrium (D′ = 1.0) and showed the strongest effect on growth and muscle mass (LOD ≥ 4.00). This reduced region contains 30 genes, compared to 292 genes in the original region. Chicken 60 K and 600 K SNP chips combined with DNA sequencing of the parental lines were used to call mutations in the reduced region. In the narrowed‐down region, 489 sequence variants were detected between NHI and WL77. The most deleterious variants are a missense variant in ADGRA3 (SIFT = 0.02) and a frameshift deletion in the functional unknown gene ENSGALG00000014401 in NHI chicken. In addition, five synonymous variants were discovered in genes PPARGC1A, ADGRA3, PACRGL, SLIT2 and FAM184B. In our study, the confidence interval and the number of potential genes could be reduced 8‐ and 10‐ fold respectively. Further research will focus on functional effects of mutant genes.     

Receptor-mediated interference mechanism responsible for resistance to polytropic leukemia viruses in Mus castaneus

The Asian mouse Mus castaneus is resistant to infection by the polytropic mink cell focus-inducing (MCF) subgroup of murine leukemia viruses (MuLVs). Genetic crosses showed this recessive resistance to be governed by a single gene that maps at or near the gene encoding the polytropic viral receptor, Rmc1. To investigate this resistance, we mated M. castaneus with mice carrying the wild mouse Sxv variant of the Rmc1 receptor that allows infection by xenotropic as well as polytropic virus. Unlike other F1 hybrids of M. castaneus, these F1 mice were resistant to both xenotropic and polytropic classes of MuLVs. Analysis of backcrossed progeny of the F1 hybrids mated to Sxv mice indicates that resistance is due to inheritance of two M. castaneus genes. Cells from individual backcross mice were also examined for cell surface antigen by fluorescence-activated cell sorter analysis with monoclonal antibodies reactive with xenotropic or MCF virus env glycoproteins. A correlation was observed between virus resistance and antigen, suggesting that virus resistance is due to expression of endogenous viral envelope genes that interfere with infection by exogenous virus. Since the inbred strain Rmc1 receptor remains functional in the presence of these M. castaneus genes, and since M. castaneus contains multiple copies of xenotropic MuLV env genes, we suggest that these resistance genes control expression of xenotropic env glycoprotein that interferes with exogenous virus in cells containing the Sxv variant of Rmc1.     

Characterization of monoclonal antibody's binding kinetics using oblique-incidence reflectivity difference approach

Monoclonal antibodies (mAbs) against human proteins are the primary protein capture reagents for basic research, diagnosis, and molecular therapeutics. The 2 most important attributes of mAbs used in all of these applications are their specificity and avidity. While specificity of a mAb raised against a human protein can be readily defined based on its binding profile on a human proteome microarray, it has been a challenge to determine avidity values for mAbs in a high-throughput and cost-effective fashion. To undertake this challenge, we employed the oblique-incidence reflectivity difference (OIRD) platform to characterize mAbs in a protein microarray format. We first systematically determined the K_on and K_off values of 50 mAbs measured with the OIRD method and deduced the avidity values. Second, we established a multiplexed approach that simultaneously measured avidity values of a mixture of 9 mono-specific mAbs that do not cross-react to the antigens. Third, we demonstrated that avidity values of a group of mAbs could be sequentially determined using a flow-cell device. Finally, we implemented a sequential competition assay that allowed us to bin multiple mAbs that recognize the same antigens. Our study demonstrated that OIRD offers a high-throughput and cost-effective platform for characterization of the binding kinetics of mAbs.