Protein Disulfide Isomerase and Its Potential Function on Endoplasmic Reticulum Quality Control in Diatom Phaeodactylum tricornutum

PDI is a molecular chaperone and plays an important role in Endoplasmic Reticulum quality control (ERQC).PDI participates in the refolding of the misfolded/unfolded proteins to maintain cellular homeostasis under differentstresses. However, bioinformatic characteristics and potential functions of PDIs in diatom Phaeodactylumtricornutum (Pt) are still unknown so far. Hence, the genome-wide characteristics of PtPDI proteins in P. tricornutumwere first studied via bioinformatic and transcriptomic methods. 42 PtPDI genes were identified from thegenome of P. tricornutum. The motif, protein structure, classification, number of introns, phylogenetic relationship,and the expression level of 42 PtPDI genes under the tunicamycin stress were analyzed. A pair of tandemduplicated genes (PtPDI15 and PtPDI18) was observed in P. tricornutum. The 42 PtPDIs with different genecharacteristics were divided into three independent clades, indicating different evolutional relationships and functionsof these PtPDIs. The 14 up-regulated PtPDI genes under the tunicamycin treatment might have a positiveeffect on the ER quality control of the unfolded/misfolded proteins, while the 7 down-regulated PtPDIs mightnegatively affect the ERQC. The characteristics of all 42 PtPDIs and their proposed working model here providea comprehensive understanding of the PtPDIs gene family. The differential expression of 21 PtPDIs will be usefulfor further functional study in the ERQC.

Process Control and Optimization for Heterologous Protein Production by Methylotrophic Pichia pastoris

The methylotrophic yeast Pichia pastoris is a highly successful system for production of a variety of heterologous proteins due to its unique features/abilities for effective protein expression, and tremendous efforts have been made to increase heterologous protein productivity by P. pastoris in recent years. When new engineered yeast strains are constructed and are ready to use tot industrial protein production, process control and optimization techniques should be applied to improve the fermentation performance in the following aspects： （1） increase recombinant cell concentrations in fermentor to high density during growth phase; （2） effectively induce heterologous proteins by enhancing/stabilizing titers or concentrations of the proteins during induction phase; （3） decrease operation costs by relieving the working loads of heat-exchange and oxygen supply. This article reviews and discusses the key and commonly used techniques in heterologous protein production by P. pastoris, with the focus on optimizations of fermentation media and basic operation conditions, development of optimal glycerol feeding strategies for achieving high density cultivation of P. pastoris and effective heterologous protein induction methods by regulating specific growth rate, methanol concentration, temperatures, mixture ratio of multi-carbon substrates, etc. Metabolic analysis for recombinant protein production by P. pastoris is also introduced to interpret the mechanism of sub-optimal heterologous protein production and to explore further optimal expression methods.

cDNA cloning and mRNA expression of the translationally controlled tumor protein(TCTP)gene from Japanese sea perch(Lateolabrax japonicus)

A homologue of the lower vertebrates translationally controlled tumor protein (TCTP) was cloned from the marine fish Japanese sea perch (Lateolabrax japonicus) by the technology of homology cloning. The full-length cDNA sequence of the sea perch TCTP gene contained a 5' untranslated region (UTR) of 47 bp, a 3' UTR of 433 bp, and a putative open reading frame (ORF) of 510 bp encoding a polypeptide of 170 amino acids. The deduced amino acid sequence of the sea perch TCTP gene showed a high similarity to that of zebrafish, rohu, rabbit, chicken and human. Sequence analysis revealed there were a signature sequence of TCTP family, an N-glycosylation site, and five Casein kinase phosphorylation sites in the sea perch TCTP. The temporal expression of TCTP genes in healthy and lipopolysaccharide (LPS) challenged fishes was measured by semi-quantitative reverse transcription-PCR (RT-PCR). The results indicated that LPS could up-regulate the expression of sea perch TCTP in the examined tissues, including head-kidney, spleen and liver.

The Life of a Protein Molecule——Protein Quality Control

The research progress in molecular chaperones, unfolded protein response （UPR） and ER-associated degradation （ERAD） involved in the protein quality control was summarized in this paper, and then the existing problems and the future devel- opment prospect were also discussed. It was pointed out that the life process of protein experienced four stages including synthesizing, folding, assembling and degradation, while each stage required strict quality control. In endoplasmic reticulum （ER）, a variety of proteins had been synthesized, folded and modified to form func- tional proteins with certain conformation. When the folding was blocked in ER, the unfolded proteins would aggregate and induce the UPR, which up-regulated the level of modification enzymes folded by a series of molecular chaperones and proteins to help them accomplish folding and assembling. If these proteins were still folded incorrectly, they would enter into ERAD for being degraded.

Controllable fabrication of nitrogen-doped porous nanocarbons for high-performance supercapacitors via supramolecular modulation strategy

In the present work,we developed a micellar system of milk protein-surfactant(SDS)-graphene to prepare the graphene-based aerogels via hydrothermal and freeze-drying method,in which the novel surface-property of aerogels can be tuned with the decreasing of micellar size in the colloid systems resulting the improved specific surface area.The milk protein also severed as green and sustainable sources to introduce nitrogen heteroatoms into the aerogels.Subsequently,the aerogels were further graphitized and activated to fabricate N-doped porous nanocarbon at 600℃.The initial surface composition and structure of the aerogel,which was proved,has obvious impact on the final structure of the synthesized nanocarbon materials,and thus influence their electrochemical activity.The optimized nanocarbon materials(MGPC-5),with enhanced specific surface area,degree of graphitization,and nitrogen doping,exhibited excellent capacitance performance and stability in both three-electrode system(518.8 F/g at a current density of 0.1 A/g)and symmetrical electrode system(120.8 F/g at current density of 0.1 A/g and with^95%capacitance retention after 5000 cycles of charging and discharging at 3 A/g)in KOH.The assembled supercapacitor also shows ideal capacitive properties in series and parallel configurations.Tested with a stable 1.6 V windows in Li2SO4 electrolyte,the symmetric supercapacitor cell exhibits a high energy density up to 36.7 W h/kg.The present work provides a feasible fabrication method for high-performance supercapacitor based on graphene and biomass derived carbon,the proposed surfaceproperty regulation and supercapacitor performance improvement strategy may also shed light on other energy related materials or system.

Recent advances in crystalline and amorphous particulate protein formulations for controlled delivery

The number of particulate delivery systems for biologics is negligible compared to liquid dosage forms, signifying the complications associated with development of solid protein delivery systems. Particulate protein delivery systems can improve stability, reduce viscosity of suspensions at high protein concentration and allow for controlled drug release. This review discusses current advances in controlled delivery of particulate protein formulations. While the focus lies on protein crystals and delivery systems employing protein crystals,amorphous protein particles will also be addressed. Crystallization and precipitations methods and modifications allowing controlled delivery with and without encapsulation are summarized and discussed.

Cloning, Expression and Characterization of Translationally Controlled Tumor Protein (TCTP) Gene from Flatfish Turbot (Scophthalmus maximus)

A full-length cDNA encoding translationally controlled tumor protein of marine flatfish turbot （Scophthalmus maximus）, SmTCTP, was isolated with rapid amplification of cDNA Ends （RACE）. SmTCTP consisted of a 5＇ untranslated region （UTR） of 84 bp, a 3＇ UTR of 451 bp and an open reading frame （ORF） of 513 bp, encoding a protein of 170 amino acid residues, which contained two signature sequences of TCTP family. The 5＇UTR of SmTCTP started with a 5＇-terminal oligopyrimidine tract （5＇-TOP）, a typical feature for translationally controlled mRNAs. The deduced amino acid sequence of SmTCTP was similar to the other known vertebrate TCTPs in a range of 58.8% to 64.1%. The length of fish TCTPs was diverse among species, e.g., TCTP of turbot and sea perch （Lateolabraxjaponicus） is 170 aa in length, while that of zebrafish （Danio rerio） and rohu （Labeo rohita） is 171 aa in length. Northern blot analysis revealed that SmTCTP has only one type of mRNA. Its expression level in albino skin was slightly higher than that in normal skin. We constructed the pET3Oa-SmTCTP expression plasmid. The recombinant protein of His-tag SmTCTP was over-expressed in E. coli, purified and identified with peptide mass fingerprinting. These results may pave the way of further investigation of the biological function of TCTP in fish.

The pathogenic mechanism of TAR DNA-binding protein 43(TDP-43)in amyotrophic lateral sclerosis

The onset of amyotrophic lateral sclerosis is usually characterized by focal death of both upper and/or lower motor neurons occurring in the motor cortex,basal ganglia,brainstem,and spinal cord,and commonly involves the muscles of the upper and/or lower extremities,and the muscles of the bulbar and/or respiratory regions.However,as the disease progresses,it affects the adjacent body regions,leading to generalized muscle weakness,occasionally along with memory,cognitive,behavioral,and language impairments;respiratory dysfunction occurs at the final stage of the disease.The disease has a complicated pathophysiology and currently,only riluzole,edaravone,and phenylbutyrate/taurursodiol are licensed to treat amyotrophic lateral sclerosis in many industrialized countries.The TAR DNA-binding protein 43 inclusions are observed in 97%of those diagnosed with amyotrophic lateral sclerosis.This review provides a preliminary overview of the potential effects of TAR DNAbinding protein 43 in the pathogenesis of amyotrophic lateral sclerosis,including the abnormalities in nucleoplasmic transport,RNA function,post-translational modification,liquid-liquid phase separation,stress granules,mitochondrial dysfunction,oxidative stress,axonal transport,protein quality control system,and non-cellular autonomous functions(e.g.,glial cell functions and prion-like propagation).

Capillary Electrophoretic Separation of Proteins under the Control of Radial Electric Field

Separation of basic proteins was performed using a homemade field-modulated capillary electrophoresis system. The resolution. elution and even wall adsorption can be regulated by ad-lusting the radial rather than axial voltage applied. Selection of running buffer and pH was found to be critical.

Advances in Research and Application of gE Gene/Protein in Prevention and Control of Swine Pseudora-bies

Research and application progresses of gE gene and its encoding gE protein in PR vaccines, diagnostic technique and epidemiological investigation are summarized, which have certain reference value for comprehensive prevention and control of PR and gradual purification of PR in different regions.

Translationally controlled tumor protein exerts a proin?ammatory role in acute rejection after liver transplantation

Background:Translationally controlled tumor protein(TCTP),which has been verified to have a proinflammatory activity,plays an important role in allergy.However,it remains unclear whether TCTP has an impact on the acute rejection(AR)after liver transplantation.Methods:Three protocols were used to delineate the role of TCTP in AR after liver transplantation.First,in rat orthotopic liver transplantation(OLT),the expression of TCTP was measured by enzyme-linked immunosorbent assay(ELISA),real-time PCR,Western blot and immunofluorescence assays.Second,in mixed lymphocyte reaction(MLR),the role of TCTP in lymphocyte proliferation was measured by carboxyfluorescein succinimidyl ester(CFSE)labeling and the impact of TCTP on inflammatory factor release was detected by cytokine arrays.Third,in human OLT,the level of serum TCTP was detected by ELISA,and the relationship between TCTP and model for early allograft function(MEAF)score was assessed by Spearman's correlation.Results:In rat OLT,AR resulted in great harm to allografts,manifesting as deterioration of liver function,increasing inflammatory factors and infiltrating lymphocytes.Meanwhile,TCTP was overexpressed in serum and allografts.Higher level of TCTP was associated with higher rejection activity index(RAI).In an MLR protocol,TCTP knockdown inhibited the proliferation of mixed inflammatory cells and significantly suppressed the release of 15 cytokines and chemokines.In human OLT,the serum TCTP was up-regulated within a week after operation.Additionally,the increasing speed of serum TCTP positively correlated with MEAF scores(r=0.449;P=0.0088).

A new size and shape controlling method for producing calcium alginate beads with immobilized proteins

A method for producing size- and shape-con-trolled calcium alginate beads with immobilized proteins was developed. Unlike previous cal-cium alginate bead production methods, pro-tein-immobilized alginate beads with uniform shape and sizes less then 20 micrometers in diameter could successfully be produced by using sonic vibration. BSA and FITC-conjugated anti-BSA antibodies were used to confirm pro-tein immobilization in the alginate beads. Pro-tein diffusion from the beads could be reduced to less than 10% by cross-linking the proteins to the alginate with 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC) and N-hydroxysul-fosuccinimide (NHSS). The calcium alginate beads could also be arranged freely on a slide glass by using a femtosecond laser.

Dual-functional poly(2-methyl-2-oxazoline)/poly(2-(dimethylamine)ethyl methacrylate)mixed brushes with switchable protein adsorption and antibacterial properties

Herein,binary mixed brushes consisting of poly(2-methyl-2-oxazoline)(PMOXA)and poly(2-(dimethylamine)ethyl methacrylate)(PDMAEMA)with different chain lengths were fabricated by successive grafting of NH_(2)-terminated PMOXA and SH-terminated PDMAEMA onto polydopamine-anchored substrates.The mixed-brush coating was characterized by variable-angle spectroscopic ellipsometry,X-ray photoelectron spectroscopy,Fourier transform infrared spectroscopy,zeta potential measurements,water contact angle,and atomic force microscopy.The mixed brushes showed tunable surface charge,wettability,and surface roughness,depending on the degree of PDMAEMA swelling under varying pH and ionic strength(Ⅰ).Then the adsorption behaviors of pepsin,bovine serum albumin(BSA),γ-globulin,and lysozyme,four very different proteins with regard to isoelectric point,on the mixed brushes coating were studied by using fluorescence microscopy and surface plasmon resonance.When the chain length of PDMAEMA was about twice as long as PMOXA,the mixed brushes not only had high adsorption capacity for pepsin,BSA,and y-globulin but also had a desorption efficiency of 86.9%,87.1%,and 93.5%,respectively.It is explained that electrostatic attraction between the protonated PDMAEMA and positively charged acidic proteins(pepsin and BSA,whose isoelectric points were below the pK_(a) of PDMAEMA)would drive the intensive adsorption(at pH 3,I=10^(-3)mol·L^(-1)for pepsin,and pH 5,I=10^(-5)mol·L^(-1)for BSA),while desorption was dominated by the hydrophilic PMOXA when PDMAEMA was shrinking(at pH 7,I=10^(-1)mol·L^(-1)for pepsin,and pH 9,I=10^(-1)mol·L^(-1)for BSA).Furthermore,the isoelectric precipitation led to the adsorption of neutral protein(γ-globulin,whose isoelectric point was near the pK_a of PDMAEMA)at pH 7,I=10^(-5)mol·L^(-1),while electrostatic repulsion and antifouling PMOXA triggered the desorption of y-globulin at pH 3,I-10^(-1)mol·L^(-1).However,alkaline protein(lysozyme,whose isoelectric point was higher than the pK_(a) of PDMAEMA)exhibited slight adsorption on PMOXA/PDMAEMA mixed brushes under test conditions,regardless of whether PMOXA or PDMAEMA occupied the outermost layer.The antibacterial property of the mixed brushes against Escherichia coli was investigated.PMOXA/PDMAEMA mixed brushes showed significant bactericidal activity at pH 3,I=10^(-3)mol·L^(-1),while the rinse of pH 9,I=10^(-1)mol·L^(-1)solution could remove most of the residual bacteria.This work not only enables controlled adsorption of proteins with different isoelectric points but also ensures that the surface of the coating is minimized from bacterial contamination.

Basic regulatory science behind drug substance and drug product specifications of monoclonal antibodies and other protein therapeutics

In this review,we focus on providing basics and examples for each component of the protein therapeutic specifications to interested pharmacists and biopharmaceutical scientists with a goal to strengthen understanding in regulatory science and compliance.Pharmaceutical specifications comprise a list of important quality attributes for testing,references to use for test procedures,and appropriate acceptance criteria for the tests,and they are set up to ensure that when a drug product is administered to a patient,its intended therapeutic benefits and safety can be rendered appropriately.Conformance of drug substance or drug product to the specifications is achieved by testing an article according to the listed tests and analytical methods and obtaining test results that meet the acceptance criteria.Quality attributes are chosen to be tested based on their quality risk,and consideration should be given to the merit of the analytical methods which are associated with the acceptance criteria of the specifications.Acceptance criteria are set forth primarily based on efficacy and safety profiles,with an increasing attention noted for patient-centric specifications.Discussed in this work are related guidelines that support the biopharmaceutical specification setting,how to set the acceptance criteria,and examples of the quality attributes and the analytical methods from 60 articles and 23 pharmacopeial monographs.Outlooks are also explored on process analytical technologies and other orthogonal tools which are on-trend in biopharmaceutical characterization and quality control.

Regulation and function of endoplasmic reticulum autophagy in neurodegenerative diseases

The endoplasmic reticulum,a key cellular organelle,regulates a wide variety of cellular activities.Endoplasmic reticulum autophagy,one of the quality control systems of the endoplasmic reticulum,plays a pivotal role in maintaining endoplasmic reticulum homeostasis by controlling endoplasmic reticulum turnover,remodeling,and proteostasis.In this review,we briefly describe the endoplasmic reticulum quality control system,and subsequently focus on the role of endoplasmic reticulum autophagy,emphasizing the spatial and temporal mechanisms underlying the regulation of endoplasmic reticulum autophagy according to cellular requirements.We also summarize the evidence relating to how defective or abnormal endoplasmic reticulum autophagy contributes to the pathogenesis of neurodegenerative diseases.In summary,this review highlights the mechanisms associated with the regulation of endoplasmic reticulum autophagy and how they influence the pathophysiology of degenerative nerve disorders.This review would help researchers to understand the roles and regulatory mechanisms of endoplasmic reticulum-phagy in neurodegenerative disorders.

基于ControlLogix系统的大豆分离蛋白过程网络控制系统

本文介绍了基于Rockwell公司的ControlLogix控制系统而研制开发的大豆分离蛋白过程网络控制系统 ,介绍了控制系统的硬件构成 。

Era蛋白(E.coli ras-like protein)——一个可能参与真、原核细胞信号调控的新分子开关

充足的证据表明:G蛋白作为真核细胞的重要分子开关参与细胞的增殖调控以及部分代谢调控过程。而近年来在大肠杆菌中新发现的Era蛋白(E.coli ras-like protein)则是与已知的三聚体G蛋白和小分子G蛋白不同的一种新的GTP结合蛋白。进一步的研究发现该类GTP结合蛋白不仅存在于原核的大肠杆菌中,而且在高等植物、人类细胞中均含有该蛋白的同源蛋白。大肠杆菌的Era蛋白主要位于细胞膜的内侧,在细胞质中也有一定的分布;真核细胞ERA(ERG)蛋白来源于原核细胞,定位于线粒体或叶绿体上。ERA或ERG蛋白有可能担负着与其它两类G蛋白同样重要的分子开关功能。已有的研究表明,Era蛋白参与调节原核生物的细胞分裂、细胞周期以及部分细胞代谢过程;在哺乳动物细胞中,ERA的同源蛋白可能与细胞周期的G1期调控以及细胞凋亡有关;真核植物中相关研究报道尚少,推测该蛋白可能与种子的正常发育有关。

Era蛋白(E.coli ras-like protein)-一个可能参与真、原细胞信号调控的新分子开关

充足的证据表明G蛋白作为真核细胞的重要分子开关参与细胞的增殖调控以及部分代谢调控过程。而近来在大肠杆菌中新发现的Era蛋白(E.coli ras-like protein)则是与已知的三聚体G蛋白和小分子G蛋白不同的一种新的GTP结合蛋白。进一步的研究发现该类GTP结合蛋白不仅存在于原核的大肠杆菌中,而且在高等植物、人类细胞中均含有该蛋白的同源蛋白。大肠杆菌的Era蛋白主要位于细胞膜的内侧,细胞质中也有一定的分布;一些证据表明,真核细胞ERA(ERG)蛋白来源于原核细胞,定位于线粒体或者叶绿体。近来的研究证据表明ERA或者ERG蛋白有可能担负着与其它两类G蛋白同样重要的分子开关功能。已有的研究表明Era蛋白参与调节原核生物的细胞分裂、细胞周期以及部分细胞代谢过程;在哺乳动物细胞中,同源蛋白ERA可能与细胞周期的G1期调控以及细胞凋亡有关;真核植物中相关研究报道尚少,推测该蛋白可能与种子的正常发育有关。本文主要介绍原核Era蛋白和真核ERA蛋白的结构特点以及功能研究进展。

Chitosan nanoparticles crosslinked by glycidoxypropyltrimethoxysilane for pH triggered release of protein

pH-responsive-chitosan nanoparticles for the control release of protein drug were prepared by combining two-step crosslinking method, in which chitosan was subsequently crosslinked by sodium tripolyphosphate （TPP） and glycidoxypropyltrimethoxysilane （GPTMS）. Compared with TPP crosslinked chitosan particles, the two-step crosslinked nanoparticles were not only pH-responsive but also more stable in wide pH range. Fluorescein isothiocyanate （FITC） labeled anti-human-IgG antibody was used as a model protein drug for evaluating the control release profile of the nano-carrier. The amount of released antibody increased from 5.6% to 50% when the pH of solution shifted from 7.4 to 6.0. The results suggest the possible application of the nanoparticles as pH- responsive drug delivery materials.