Discontinuous native protein gel electrophoresis: pros and cons

A new native protein gel system was recently developed that enables the rapid and convenient analysis of virtually all soluble proteins, in particular including basic proteins, in their native oligomeric states. This gel system combines the addition of negative charges to the proteins by the dye SERVA Blue G with a Tris-histidine discontinuous buffer system and the use of polyacrylamide gradient gels. The use of histidine for sample focusing rather than glycine as a slow dipolar ion following from the cathode buffer serves to improve migration of basic proteins. In this review, the principle of function as well as the advantages and disadvantages of the new gel system are discussed in the context of other native protein gel systems and further methods for the analysis of the oligomeric state of a protein.     

The protein-tethered lipid bilayer: a novel mimic of the biological membrane

A new concept of solid-supported tethered bilayer lipid membrane (tBLM) for the functional incorporation of membrane proteins is introduced. The incorporated protein itself acts as the tethering molecule resulting in a versatile system in which the protein determines the characteristics of the submembraneous space. This architecture is achieved through a metal chelating surface, to which histidine-tagged (His-tagged) membrane proteins are able to bind in a reversible manner. The tethered bilayer lipid membrane is generated by substitution of protein-bound detergent molecules with lipids using in-situ dialysis or adsorption. The system is characterized by surface plasmon resonance, quartz crystal microbalance, and electrochemical impedance spectroscopy. His-tagged cytochrome c oxidase (CcO) is used as a model protein in this study. However, the new system should be applicable to all recombinant membrane proteins bearing a terminal His-tag. In particular, combination of surface immobilization and membrane reconstitution opens new prospects for the investigation of functional membrane proteins by various surface-sensitive techniques under a defined electric field.     

Multiplexed fluorescence detection of phosphorylation, glycosylation, and total protein in the proteomic analysis of breast cancer refractoriness

The Multiplexed Proteomics (MP) technology is a new approach that permits quantitative, multicolor fluorescence detection of proteins in one-dimensional or two-dimensional gels. This methodology allows for multiplexed identification and differential analysis of phosphoproteins, glycoproteins, and total proteins within a single gel electrophoresis experiment. Here the MP system was applied to the differential proteomic analysis of pregnancy-induced refractoriness to breast cancer using a rat model system. Differential analyses identified multiple proteins with altered phosphorylation, glycosylation, or protein expression patterns.     

Finding one of a kind: advances in single-protein production

An ultimate goal for any protein production system is to express only the protein of interest without producing other cellular proteins. To date, there are only two established methods that will allow the successful expression of only the protein of interest: the cell-free in vitro protein synthesis system and the in vivo single-protein production (SPP) system. Although single-protein production can be achieved in cell-free systems, it is not easy to completely suppress the production of cellular proteins during the production of a protein of interest in a living cell. However, the finding of a unique sequence-specific mRNA interferase in Escherichia coli led to the development of the SPP system by converting living cells into a bioreactor that produces only a single protein of interest without producing any cellular proteins. This technology not only provides a new high expression system for proteins, but also offers a novel avenue for protein structural studies.     

Hepatitis C virus-like particle morphogenesis

Although much is known about the hepatitis C virus (HCV) genome, first cloned in 1989, little is known about HCV structure and assembly due to the lack of an efficient in vitro culture system for HCV. Using a recombinant Semliki forest virus replicon expressing genes encoding HCV structural proteins, we observed for the first time the assembly of these proteins into HCV-like particles in mammalian cells. This system opens up new possibilities for the investigation of viral morphogenesis and virus-host cell interactions.     

New fast BiFC plasmid assay system for in vivo protein-protein interactions.

In this age of massive genetic and protein information, a fast and reliable method of studying in vivo protein-protein interactions is necessary. We have developed a novel system that can overcome limitations of existing assay methods. This new method adopts two existing systems for fast analysis of diverse protein-protein interactions. For rapid, large-scale cloning, we adopted the Gateway system and developed novel destination vectors containing YFP N-terminus (YN) or YFP C-terminus (YC) to visualize protein-protein interactions in vivo using bimolecular fluorescence complementation (BiFC). Using this system, we investigated molecular interactions among the three POZ-domain regulatory proteins mAPM-1, LRF, KLHL10 that belong to a subgroup of human POZ-domain proteins, and showed that the POZ-domains of mAPM-1, LRF and KLHL10 could form both homodimers and heterodimers. This new method is a highly efficient, sensitive and specific assay method for protein-protein interaction in vivo.     

Acidic pH induces fusion of cells infected with baculovirus to form syncytia.

The enveloped baculovirus insect cell system has been used extensively for expression of recombinant proteins, including viral fusion proteins. We tested wild-type baculovirus for endogenous fusion protein activity. Syncytia formation, dye transfer, and capacitance changes were observed after incubating infected Spodoptera frugiperda cells in acidic media, consistent with fusion protein activity. Only a short acidic pulse of 10 s is needed to trigger syncytia formation. Identical results were obtained with recombinant baculovirus. This new system is convenient for studying pH activated cell-cell fusion. However, using this enveloped virus to study the mechanism of recombinant fusion proteins requires caution.     

The twin-arginine translocation system and its capability for protein secretion in biotechnological protein production

The biotechnological production of recombinant proteins is challenged by processes that decrease the yield, such as protease action, aggregation, or misfolding. Today, the variation of strains and vector systems or the modulation of inducible promoter activities is commonly used to optimize expression systems. Alternatively, aggregation to inclusion bodies may be a desired starting point for protein isolation and refolding. The discovery of the twin-arginine translocation (Tat) system for folded proteins now opens new perspectives because in most cases, the Tat machinery does not allow the passage of unfolded proteins. This feature of the Tat system can be exploited for biotechnological purposes, as expression systems may be developed that ensure a virtually complete folding of a recombinant protein before purification. This review focuses on the characteristics that make recombinant Tat systems attractive for biotechnology and discusses problems and possible solutions for an efficient translocation of folded proteins.     

Physiological responses to protein aggregates: Fibrinolysis, coagulation and inflammation (new roles for old factors)

Misfolding is an inherent and potentially problematic propensity of proteins. Misfolded proteins tend to aggregate and the deposition of aggregated proteins is associated with a variety of highly debilitating diseases known as amyloidoses. Protein misfolding and aggregation is also increasingly recognized as the underlying cause of other health problems, including atherosclerosis and immunogenicity of biopharmaceuticals. This raises the question how nature deals with the removal of obsolete proteins in order to avoid their accumulation and disease. In recent years two proteases, tPA and factor XII, have been identified that specifically recognize aggregates of misfolded proteins. We here review these discoveries that have uncovered new roles for the fibrinolytic system and the contact activation system beyond haemostasis.     

Minimal nanodisc without exogenous lipids for stabilizing membrane proteins in detergent-free buffer

Membrane protein stabilization after detergent solubilization presents drawbacks for structural and biophysical studies, in particular that of a reduced stability in detergent micelles. Therefore, alternative methods are required for efficient stabilization. Lipid nanodisc made with the membrane scaffold protein MSP is a valuable system but requires a fine optimization of the lipid to protein ratio. We present here the use of the scaffold protein MSP without added lipids as a minimal system to stabilize membrane proteins. We show that this method is applicable to α-helical and β-strands transmembrane proteins. This method allowed cryo-electron microscopy structural study of the bacterial transporter MexB. A protein quantification indicates that MexB is stabilized by two MSP proteins. This simplified and efficient method proposes a new advance in harnessing the MSP potential to stabilize membrane proteins.     

The resolution of membrane proteins based upon size,charge, and hydrophobicity

Currently available systems for resolving membrane proteins are based only on size and charge differences. Recently, it has been shown that Triton-urea-acetic acid gels which separate proteins on the basis of charge, size and hydrophobicity are capable of resolving proteins differing only by the substitution of a single neutral amino acid. We have applied this new method to the resolution of bacterial envelope proteins. Conditions for optimal resolution of different bacterial envelope proteins were determined by electrophoresis through transverse urea and Triton X-100 gradient gels. We have also correlated the components resolved in this system with those resolved by classical sodium dodecyl sulfate-gel electrophoresis by using two-dimensional slab gels combining the two systems. Furthermore, envelope protein fractions from different species and strains of bacteria were compared to identify specific proteins. This system appears to be a promising method for investigating envelope proteins which are due to missense mutations.     

Rcs双组分调节系统对细菌环境应答的分子调控研究进展

荚膜异多糖酸合成调节(Regulator of Capsule Synthesis,Rcs)系统是存在于许多肠杆菌科细菌中非典型的双组分调节系统,由3种核心蛋白(跨膜感应激酶RcsC、跨膜蛋白RcsD和响应调节剂RcsB)及多种辅助蛋白共同构成.Rcs系统能整合环境信号、调节基因表达并改变细菌的生理行为.近年来,对细菌...     

Human cells: new platform for recombinant therapeutic protein production

The demand for recombinant therapeutic proteins is significantly increasing. There is a constant need to improve the existing expression systems, and also developing novel approaches to face the therapeutic proteins demands. Human cell lines have emerged as a new and powerful alternative for the production of human therapeutic proteins because this expression system is expected to produce recombinant proteins with post translation modifications more similar to their natural counterpart and reduce the potential immunogenic reactions against nonhuman epitopes. Currently, little information about the cultivation of human cells for the production of biopharmaceuticals is available. These cells have shown efficient production in laboratory scale and represent an important tool for the pharmaceutical industry. This review presents the cell lines available for large-scale recombinant proteins production and evaluates critically the advantages of this expression system in comparison with other expression systems for recombinant therapeutic protein production.     

泛素样蛋白ISG15抗病毒机制研究进展

ISG15(Interferon stimulated gene 15,ISG15)蛋白是由干扰素诱导产生的一种泛素样蛋白分子,分子量大小约为15kD。ISG15同泛素分子相类似可以被共价结合于其他蛋白分子上,这种现象称为ISG化(ISGylation)现象。ISG化系统包括ISG15、UBE1L、UBCH8和HERC5四类蛋白分子,协同完成ISG化过程。ISG15及ISG化系统在抗病毒反应中具有重要作用。近几年对于ISG15的抗病毒作用和机制的研究已经有了很大的突破,ISG15的抗病毒作用也越来越受到人们重视,了解清楚ISG15抗病毒机制对于研制新的抗病毒药物及提出新的抗病毒策略具有重要意义。本文对ISG15在不同种病毒中的抗病毒机制研究进展进行了简要综述。     

Anchoring proteins to Escherichia coli cell membranes using hydrophobic anchors derived from a Bacillus subtilis integral membrane protein

Anchored periplasmic expression (APEx) technology aims to express and localize proteins or peptides in the Escherichia coli periplasm. Some reports have suggested that transmembrane segments of integral membrane proteins can be used as membrane anchors in the APEx system. In this study, a series of hydrophobic anchors derived from the first putative transmembrane helix of a Bacillus subtilis integral membrane protein, MrpF, and its truncated forms were investigated for anchored periplasmic expression of alkaline phosphatase (PhoA) in E. coli. Anchoring efficiency of hydrophobic anchors was evaluated by monitoring the expression and activity of anchored PhoA. The length of hydrophobic anchors was found to be critical for anchoring proteins to cell membranes. This study may open new avenues for applying transmembrane segments derived from native membrane proteins as membrane anchors in the APEx system.     

Identification and developmental expression of new biomineralization proteins in the sea urchin Strongylocentrotus purpuratus

The endoskeleton of the sea urchin larva is a network of calcareous rods secreted by primary mesenchyme cells (PMCs). In this study, we identified seven new biomineralization-related proteins through an analysis of a large database of gene products expressed by PMCs. The proteins include three new spicule matrix proteins (SpSM29, SpSM32, and SpC-lectin), two proteins related to the PMC-specific cell surface glycoprotein MSP130 (MSP130-related-1 and -2), and two novel proteins (SpP16 and SpP19). The genes encoding these proteins are expressed specifically by cells of the large micromere-PMC lineage and are activated zygotically beginning at the blastula stage, prior to PMC ingression. Several of the mRNAs show regulated patterns of expression within the PMC syncytium that correlate with the pattern of skeletal rod growth. This work identifies new proteins that may regulate the process of biomineralization in this tractable model system.     

New consensus nomenclature for mammalian keratins

Keratins are intermediate filament-forming proteins that provide mechanical support and fulfill a variety of additional functions in epithelial cells. In 1982, a nomenclature was devised to name the keratin proteins that were known at that point. The systematic sequencing of the human genome in recent years uncovered the existence of several novel keratin genes and their encoded proteins. Their naming could not be adequately handled in the context of the original system. We propose a new consensus nomenclature for keratin genes and proteins that relies upon and extends the 1982 system and adheres to the guidelines issued by the Human and Mouse Genome Nomenclature Committees. This revised nomenclature accommodates functional genes and pseudogenes, and although designed specifically for the full complement of human keratins, it offers the flexibility needed to incorporate additional keratins from other mammalian species.     

Alterations of gene expression in Novikoff hepatoma cells induced by a factor in human urine

Proteins and protein subunits from Novikoff hepatoma cells have been mapped by two-dimensional polyacrylamide gel electrophoresis utilizing the BASO-DALT system to resolve the basic proteins. Utilizing this technique, it has been demonstrated that human urine contains proteins that retain biological activity and can stimulate synthesis of several new proteins in neoplastic cells. This stimulatory activity has been detected in urine from cancer patients and normal individuals.