Kinetic study on starch hydrolysis using a glucose/maltose sensing system

Summary A dual-enzyme electrode flow injection system that can simultaneously determine glucose and maltose is used for an on-line study of starch hydrolyses catalysed by amylases. With the working system, determinations can be made every 2 minutes. A 10 L sample size with recycled back-flow minimises any loss of the reaction medium. The production, growth and decay of glucose and maltose concentrations during starch hydrolysis under various enzymatic conditions can thus be closely monitored, making it useful for the study of the catalytic kinetics of amylases and in screening and analysing enzyme systems.     

Sustainability of SARS-CoV-2 Induced Humoral Immune Responses in COVID-19 Patients from Hospitalization to Convalescence Over Six Months

Virologica Sinica - Understanding the persistence of antibody in convalescent COVID-19 patients may help to answer the current major concerns such as the risk of reinfection, the protection period...     

Genetically encoded redox biosensor system for H2O2 measurement in four subcellular compartments in acute myeloid leukemia (AML) cells

Science China Life Sciences -     

Studying bona fide SARS-CoV-2 biology in a BSL-2 biosafety environment using a split-virus-genome system

Science China Life Sciences -     

Identification of novel 14-3-3ζ interacting proteins by quantitative immunoprecipitation combined with knockdown (QUICK)

The family of 14-3-3 proteins has emerged as critical regulators of diverse cellular responses under both physiological and pathological conditions. To gain insight into the molecular action of 14-3-3ζ in multiple myeloma (MM), we performed a systematic proteomic analysis of 14-3-3ζ-associated proteins. This analysis, recently developed by Matthias Mann, termed quantitative immunoprecipitation combined with knockdown (QUICK), integrates RNAi, SILAC, immunoprecipitation, and quantitative MS technologies. Quantitative mass spectrometry analysis allowed us to distinguish 14-3-3ζ-interacting proteins from background proteins, resulting in the identification of 292 proteins in total with 95 novel interactions. Three 14-3-3ζ-interacting proteins-BAX, HSP70, and BAG3-were further confirmed by reciprocal coimmunoprecipitations and colocalization analysis. Our results therefore not only uncover a large number of novel 14-3-3ζ-associated proteins that possess a variety of cellular functions, but also provide new research directions for the study of the functions of 14-3-3ζ. This study also demonstrated that QUICK is a useful approach to detect specific protein-protein interactions with very high confidence and may have a wide range of applications in the investigation of protein complex interaction networks.     

Construction of a high sensitive Escherichia coli alkaline phosphatase reporter system for screening affinity peptides

An enzyme-linker-peptide fusion protein reporter system was constructed for sensitive analysis of affinity of peptide ligands to their receptor. An E. coli alkaline phosphatase (EAP) mutant enzyme with high catalytic activity was selected as the reporter protein. Interaction of affinity peptide and streptavidin was applied as demonstration of the method. Three affinity peptides, strep-tag I (SI), strep-tag II (SII) and streptavidin binding peptide (SBP) were genetically fused to the C-terminal of EAP respectively, with an insertion of a flexible linker peptide in between. The enzyme activity of the EAP fusions showed no obvious change. After expression and purification, the EAP-affinity peptide fusions were applied to the streptavidin modified surface. Binding of the fusions to the surface through interaction of affinity peptides to streptavidin was indicated by color generated from conversion of the substrate by EAP. The relative affinity and specificity of each affinity peptides to the immobilized streptavidin were then evaluated with high sensitivity and broad detection range. This method may be used for effective high-throughput screening of high affinity peptide from the peptide pool.     

A Sir2-like protein participates in mycobacterial NHEJ

In eukaryotic cells, repair of DNA double-strand breaks (DSBs) by the nonhomologous end-joining (NHEJ) pathway is critical for genome stability. In contrast to the complex eukaryotic repair system, bacterial NHEJ apparatus consists of only two proteins, Ku and a multifunctional DNA ligase (LigD), whose functional mechanism has not been fully clarified. We show here for the first time that Sir2 is involved in the mycobacterial NHEJ repair pathway. Here, using tandem affinity purification (TAP) screening, we have identified an NAD-dependent deacetylase in mycobacteria which is a homologue of the eukaryotic Sir2 protein and interacts directly with Ku. Results from an in vitro glutathione S-transferase (GST) pull-down assay suggest that Sir2 interacts directly with LigD. Plasmid-based end-joining assays revealed that the efficiency of DSB repair in a sir2 deletion mutant was reduced 2-fold. Moreover, the Δsir2 strain was about 10-fold more sensitive to ionizing radiation (IR) in the stationary phase than the wild-type. Our results suggest that Sir2 may function closely together with Ku and LigD in the nonhomologous end-joining pathway in mycobacteria.     

Construction and characterization of different MutS fusion proteins as recognition elements of DNA chip for detection of DNA mutations

Three MutS fusion systems were designed as the mutation recognition and signal elements of DNA chips for detection of DNA mutations. The expression vectors containing the encoding sequences of three recombinant proteins, Trx-His6-GFP-(Ser-Gly)6-MutS (THGLM), Trx-His6-(Ser-Gly)6-Strep tagII-(Ser-Gly)6-MutS (THLSLM) and Trx-His6-(Ser-Gly)6-MutS (THLM), were constructed by gene slicing in vitro. THGLM, THLSLM and THLM were then expressed in Escherichia coli AD494(DE3), respectively. SDS-PAGE analysis revealed that each of the expected proteins was approximately 30% of the total bacterial proteins. The recombinant proteins were purified to the purity over 90% by immobilized metal (Co2+) chelation affinity chromatography. Bioactivity assay indicated that three fusion proteins retained the mismatch-binding activity and the functions of other fusion partners. DNA chips arrayed both mismatched and unpaired DNA oligonucleotides as well as rpoB gene from Mycobacterium tuberculosis were prepared. THGLM, THLSLM and THLM that was labeled with Fluorolinktrade mark Cy3 reactive dye, were then used as both mutation recognition and labeling elements of DNA chips. The resulting DNA chips were used to detect the mismatched and unpaired mutations in the synthesized oligonucleotides and single base mutation in rpoB gene of M. tuberculosis that is resistant to rifamycin.     

Functionally orthologous viral and cellular microRNAs studied by a novel dual-fluorescent reporter system

Recent research raised the possibility that some viral microRNAs (miRNAs) may function as orthologs of cellular miRNAs. In the present work, to study the functional orthologous relationships of viral and cellular miRNAs, we first constructed a dual-fluorescent protein reporter vector system for the easy determination of miRNA function. By expressing the miRNAs and the indicator and internal control fluorescent proteins individually from a single vector, this simple reporter system can be used for miRNA functional assays that include visualizing miRNA activity in live cells. Sequence alignments indicated that the simian virus 40 (SV40) encoded miRNA sv40-mir-S1-5p contains a seed region identical to that of the human miRNA hsa-miR423-5p. Using the new reporter system, it was found that sv40-mir-S1-5p and hsa-miR423-5p downregulate the expression of common artificial target mRNAs and some predicted biological targets of hsa-miR423-5p, demonstrating that they are functional orthologs. The human immunodeficiency virus 1 (HIV-1) encoded hiv1-miR-N367 also contains a seed sequence identical to that of the human miRNA hsa-miR192. Functional assays showed that hiv1-miR-N367 and hsa-miR192 could downregulate common artificial and predicted biological targets, suggesting that these miRNAs may also act as functional orthologs. Thus, this study presents a simple and universal system for testing miRNA function and identifies two new pairs of functional orthologs, sv40-mir-S1-5p and hsa-miR423-5p as well as hiv-1-miR-N367 and hsa-miR192. These findings also expand upon our current knowledge of functional homology and imply that a more general phenomenon of orthologous relationships exists between viral and cellular miRNAs.     

DNA错配修复蛋白MutS和MutL的相互作用研究

MutL 和 MutS 是DNA错配修复系统中起关键作用的修复蛋白. 利用基因融合技术高效表达了MutL 和 MutS融合蛋白,并利用它们发展了一种研究二者相互作用的简便方法. 融合蛋白MutL-GFP (Trx-His6-GFP-(Ser-Gly)6-MutL),MutL-Strep tagⅡ (Trx-His6-(Ser-Gly)6-Strep tagⅡ-(Ser-Gly)6-MutL) 和 MutS (Trx-His6-(Ser-Gly)6-MutS) 被构建并在大肠杆菌中高效表达. 收集菌体细胞、超声波破碎后离心取上清进行SDS-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 分析,结果表明有与预期分子质量相应的诱导表达条带出现,其表达量约占全细胞蛋白的30％且以可溶形式存在. 利用固定化金属离子配体亲和层析柱分别纯化融合蛋白,其纯度达到90％. 通过将MutS蛋白固定的方法研究两种MutL融合蛋白分别与MutS之间的相互作用. 结果表明：只有MutS蛋白与含有错配碱基DNA分子结合后才与MutL蛋白发生相互作用. 通过检测MutL融合蛋白标记的绿色荧光信号或酶学显色信号来鉴定相互作用的发生. 建立的融合分子系统方法也为研究其他的蛋白质或生物大分子之间的相互作用提供了一个技术平台.