High‐throughput instant quantification of protein expression and purity based on photoactive yellow protein turn off/on label

Quantifying the concentration and purity of a target protein is essential for high‐throughput protein expression test and rapid screening of highly soluble proteins. However, conventional methods such as PAGE and dot blot assay generally involve multiple time‐consuming tasks requiring hours or do not allow instant quantification. Here, we demonstrate a new method based on the Photoactive yellow protein turn Off/On Label (POOL) system that can instantly quantify the concentration and purity of a target protein. The main idea of POOL is to use Photoactive Yellow Protein (PYP), or its miniaturized version, as a fusion partner of the target protein. The characteristic blue light absorption and the consequent yellow color of PYP is absent when initially expressed without its chromophore, but can be turned on by binding its chromophore, p‐coumaric acid. The appearance of yellow color upon adding a precursor of chromophore to the co‐expressed PYP can be used to check the expression amount of the target protein via visual inspection within a few seconds as well as to quantify its concentration and purity with the aid of a spectrometer within a few minutes. The concentrations measured by the POOL method, which usually takes a few minutes, show excellent agreement with those by the BCA Kit, which usually takes ～1 h. We demonstrate the applicability of POOL in E. coli, insect, and mammalian cells, and for high‐throughput protein expression screening.     

First attempts to crystallize a non-homogeneous sample of thioredoxin from Litopenaeus vannamei: What to do when you have diffraction data of a protein that is not the target?

The importance of sample homogeneity and purity in protein crystallization is essential to obtain high-quality diffracting crystals. Here, in an attempt to determine the crystal structure of thioredoxin 1 from whiteleg shrimp Litopenaeus vannamei (LvTrx), we inadvertently crystallized the hexameric inorganic pyrophosphatase of Escherichia coli (E-PPase) from a non-homogeneous sample product during the initial over-expression steps and partial purification of LvTrx. The structure determination and identification of the crystallized protein were derived from several clues: the failures in the Molecular Replacement (MR) trials using LvTrx coordinates as a search model, the unit cell parameters and space group determination, and essentially by the use of the program BALBES. After using the previously deposited E-PPase structure (PDB entry 1mjw) as a search model and the correct space group assignation, the MR showed an E-PPase complexed with SO₄^?2 with small changes in the sulfate ion binding region when it compares to previously deposited E-PPases in the PDB. This work stresses the importance of protein purity to avoid the risk of crystallizing a contaminant protein or how pure need to be a protein sample in order to increase the possibility to obtain crystals, but also serves as a reminder that crystallization is by itself a purification process and how the program BALBES can be useful in the crystal structure determination of previously deposited structures in the PDB.     

Bringing Conducting Polymers to High Order: Toward Conductivities beyond 105 S cm−1 and Thermoelectric Power Factors of 2 mW m−1 K−2

Here, an effective design strategy of polymer thermoelectric materials based on structural control in doped polymer semiconductors is presented. The strategy is illustrated for two archetypical polythiophenes, e.g., poly(2,5‐bis(3‐dodecyl‐2‐thienyl)thieno[3,2‐b]thiophene) (C₁₂‐PBTTT) and regioregular poly(3‐hexylthiophene) (P3HT). FeCl₃ doping of aligned films results in charge conductivities up to 2 × 10⁵ S cm^?1 and metallic‐like thermopowers similar to iodine‐doped polyacetylene. The films are almost optically transparent and show strongly polarized near‐infrared polaronic bands (dichroic ratio >10). The comparative study of structure–property correlations in P3HT and C₁₂‐PBTTT identifies three conditions to obtain conductivities beyond 10⁵ S cm^?1: i) achieve high in‐plane orientation of conjugated polymers with high persistence length; ii) ensure uniform chain oxidation of the polymer backbones by regular intercalation of dopant molecules in the polymer structure without disrupting alignment of π‐stacked layers; and iii) maintain a percolating nanomorphology along the chain direction. The highly anisotropic conducting polymer films are ideal model systems to investigate the correlations between thermopower S and charge conductivity σ. A scaling law S ∝ σ^?1/4 prevails along the chain direction, but a different S ∝ ?ln(σ) relation is observed perpendicular to the chains, suggesting different charge transport mechanisms. The simultaneous increase of charge conductivity and thermopower along the chain direction results in a substantial improvement of thermoelectric power factors up to 2 mW m^?1 K^?2 in C₁₂‐PBTTT.     

Tailoring of recombinant FDH: effect of histidine tag location on solubility and catalytic properties of Chaetomium thermophilum formate dehydrogenase (CtFDH)

Abstract

Several protein expression systems can be used to get enzymes in required quantities and study their functions. Incorporating a polyhistidine tag is a beneficial way of getting various enzymes such as FDHs for industrial applications. The NAD⁺ dependent formate dehydrogenase from Chaetomium thermophilum (CtFDH) can be utilized for interconversion of formate to carbon dioxide coupled with the conversion of NAD⁺ to NADH. In this study, N-terminal His tagged CtFDH (N-CtFDH) and C-terminal His tagged CtFDH (C-CtFDH) was constructed to learn the effect of His tag location on the activity and kinetic parameters of the enzyme. The solubility of proteins was not affected by tag position, however, an interference on the N-terminal region caused a deterioration in specific activity and the kinetic ability of enzyme. The obtained results indicated that the C-terminus of the enzyme is an appropriate region for tag engineering. The C-CtFDH has an approximately three-fold larger specific activity and two-fold higher catalytic efficiency than N-CtFDH. The results suggest that insertion of a His-tag at the N-terminal or C-terminal end of CtFDH has different effects on the protein and the N-terminal fragment of the protein is crucial for the function of CtFDH.     

Type III-A CRISPR-Cas Csm Complexes: Assembly,Periodic RNA Cleavage,DNase Activity Regulation,and Autoimmunity

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人二氢乳清酸脱氢酶蛋白的表达、纯化及其与新型抑制剂的晶体结构

人二氢乳清酸脱氢酶（human dihydroorotate dehydrogenase, hDHODH）是催化嘧啶从头合成途径的一个关键酶。近年来,多种研究表明,抑制该酶可缓解类风湿性关节炎的症状。但该酶的抑制剂甚少,寻找该酶的高效抑制剂具有重要意义。本研究利用PCR技术扩增hDHODH基因,构建重组质粒pET-19b-hDHODH,并在大肠杆菌(Escherichia coli, E.coli ) BL21(DE3)中表达,获得可溶性蛋白质。用Ni2+-NTA亲和层析柱对蛋白质进行纯化,获得较高（90%）纯度的hDHODH蛋白,将蛋白质与抑制剂3-(5-乙硫基)-1H-1, 2, 4-三氮唑-3-)苯甲酸和底物DHO混合孵育。用Hampton试剂盒初筛晶体并用棋盘法进行优化,获得晶形完美、衍射能力很强的hDHODH蛋白复合物单晶。用X射线衍射晶体,用CCP4、Coot软件解析结构,获得hDHODH蛋白复合物晶体结构。从解析的结构中可以看出,抑制剂与蛋白质的吻合度非常高,且抑制剂通过亲水的羧基端与蛋白质356位和147位的酪氨酸形成氢键网络。抑制剂的5元环与蛋白质359位的亮氨酸和360位的苏氨酸相互作用,使抑制剂与蛋白质牢固结合。该复合物晶体结构的顺利解析,将为开发新型特异性抗类风湿性关节炎药物提供重要基础。     

Migratory patterns and connectivity of two North American grassland bird species

Effective management and conservation of migratory bird populations require knowledge and incorporation of their movement patterns and space use throughout the annual cycle. To investigate the little‐known migratory patterns of two grassland bird species, we deployed 180 light‐level geolocators on Grasshopper Sparrows (Ammodramus savannarum) and 29 Argos‐GPS tags on Eastern Meadowlarks (Sturnella magna) at Konza Prairie, Kansas, USA, and six US Department of Defense (DoD) installations distributed across the species' breeding ranges. We analyzed location data from 34 light‐level geolocators and five Argos‐GPS tags attached for 1 year to Grasshopper Sparrows and Eastern Meadowlarks, respectively. Grasshopper Sparrows were present on the breeding grounds from mid‐April through early October, substantially longer than previously estimated, and migrated on average ~2,500 km over ~30 days. Grasshopper Sparrows exhibited strong migratory connectivity only at a continental scale. The North American Great Lakes region likely serves as a migratory divide for Midwest and East Coast Grasshopper Sparrows; Midwest populations (Kansas, Wisconsin, and North Dakota; n = 13) largely wintered in Texas or Mexico, whereas East Coast populations (Maryland and Massachusetts, n = 20) wintered in the northern Caribbean or Florida. Our data from Eastern Meadowlarks provided evidence for a diversity of stationary and short‐ and long‐distance migration strategies. By providing the most extensive examination of the nonbreeding movement ecology for these two North American grassland bird species to date, we refine information gaps and provide key insight for their management and conservation.     

Estimating age‐dependent survival from age‐aggregated ringing data—extending the use of historical records

Bird ring‐recovery data have been widely used to estimate demographic parameters such as survival probabilities since the mid‐20th century. However, while the total number of birds ringed each year is usually known, historical information on age at ringing is often not available. A standard ring‐recovery model, for which information on age at ringing is required, cannot be used when historical data are incomplete. We develop a new model to estimate age‐dependent survival probabilities from such historical data when age at ringing is not recorded; we call this the historical data model. This new model provides an extension to the model of Robinson, 2010, Ibis, 152, 651–795 by estimating the proportion of the ringed birds marked as juveniles as an additional parameter. We conduct a simulation study to examine the performance of the historical data model and compare it with other models including the standard and conditional ring‐recovery models. Simulation studies show that the approach of Robinson, 2010, Ibis, 152, 651–795 can cause bias in parameter estimates. In contrast, the historical data model yields similar parameter estimates to the standard model. Parameter redundancy results show that the newly developed historical data model is comparable to the standard ring‐recovery model, in terms of which parameters can be estimated, and has fewer identifiability issues than the conditional model. We illustrate the new proposed model using Blackbird and Sandwich Tern data. The new historical data model allows us to make full use of historical data and estimate the same parameters as the standard model with incomplete data, and in doing so, detect potential changes in demographic parameters further back in time.