Forsythakhtar0229

Tandem affinity purification is a useful strategy to isolate multisubunit complexes of high yield and purity but can be limited when working with halophilic proteins that are not properly expressed in Escherichia coli. Cyclopamine concentration Halophilic proteins are desirable for bioindustrial applications as they are often stable and active in organic solvents; however, these proteins can be difficult to express, fold, and purify by traditional technologies. Haloarchaea provide a useful alternative for expression of halophilic proteins. These microorganisms use a salt-in strategy to maintain homeostasis and express most of their proteins with halophilic properties and low pI. Here, we provide detailed protocols for the genetic modification, expression and tandem affinity purification of "salt-loving" multisubunit complexes from the haloarchaeon Haloferax volcanii. The strategy for isolation of affinity tagged 20S proteasomes that form cylindrical proteolytic nanomachines of α1, α2 and β subunits is described.One of the goals in recombinant protein production in Escherichia coli is to maximize productivity. High volumetric and specific yields can be reached after careful selection of expression strains and optimization of cultivation parameters. In this chapter, we review the many tools available to make the most out of this versatile microbial cell factory. Useful guidelines and options for troubleshooting production are presented.Haloarchaea and their enzymes have extremophilic properties desirable for use as platform organisms and biocatalysts in the bioindustry. These GRAS (generally regarded as safe) designated microbes thrive in hypersaline environments and use a salt-in strategy to maintain osmotic homeostasis. This unusual strategy has resulted in the evolution of most of the intracellular and extracellular enzymes of haloarchaea to be active and stable not only in high salt (2-5M) but also in low salt (0.2M). This salt tolerance is correlated with a resilience to low water activity, thus, rendering the haloarchaeal enzymes active and stable in organic solvent and temperatures of 50-60°C used in the enzymatic biodelignification and saccharification of lignocellulosic materials. High-level secretion of haloarchaeal enzymes to the extracellular milieu is useful for many applications, including enzymes that deconstruct biomass to allow for lignin depolymerization and simultaneous fermentation of sugars released from hemicellulose and cellulose fractions of lignocellulosics. Here we detail strategies and methods useful for high-level secretion of a laccase, HvLccA, that mediates oxidation of various phenolics by engineering a recombinant strain of the haloarchaeon Haloferax volcanii.Since its invention, recombinant protein expression has greatly facilitated our understanding of various cellular processes in different biological systems because theoretically this technique renders any gene to be expressed in a mesophilic host like Escherichia coli, thus allowing functional characterizations of proteins of interest. However, such a practice has only yielded a limited success for proteins encoded in thermophilic archaea since thermophilic proteins are often present in an insoluble form when expressed in E. coli. As a result, it is advantageous to express recombinant proteins of thermophilic archaea in a homologous host, allowing a native form of recombinant protein to be purified and characterized. Here we present a detailed protocol for the homologous expression and purification of proteins in the thermophilic archaeon, Sulfolobus islandicus Rey15A.Hyperthermophiles, typically defined as organisms with growth optima ≥80°C, are dominated by the Archaea. Proteins that support life at the extremes of temperatures often retain substantial biotechnological and commercial value, but the recombinant expression of individual hyperthermophilic proteins is commonly complicated in non-native mesophilic hosts due to differences in codon bias, intracellular solutes and the requirement for accessory factors that aid in folding or deposition of metal centers within archaeal proteins. The development of versatile protein expression and facilitated protein purification systems in the model, genetically tractable, hyperthermophilic marine archaeon Thermococcus kodakarensis provides an attractive platform for protein expression within the hyperthermophiles. The assortment of T. kodakarensis genetic backgrounds and compatible selection markers allow iterative genetic manipulations that facilitate protein overexpression and expedite protein purifications. Expression vectors that stably replicate both in T. kodakarensis and Escherichia coli have been validated and permit high-level ectopic gene expression from a variety of controlled and constitutive promoters. Biologically relevant protein associations can be maintained during protein purifications to identify native protein partnerships and define protein interaction networks. T. kodakarensis thus provides a versatile platform for the expression and purification of thermostable proteins.Neutron scattering is a powerful technique for determining the structure and dynamics of biological materials in a variety of environmental conditions. A distinguishing property of the neutron is its sensitivity to detecting hydrogen and distinguishing it from its isotope deuterium. This enables unique types of experiments that take advantage of this differential sensitivity called isotopic contrast variation. Using this approach, the chemistry of the system is not changed, but the visibility of individual sample components can be tuned by varying the deuterium content of the system under investigation. Deuterated proteins are commonly produced in bacterial systems that are adapted to growth in D2O minimal media. To maximize the yield of deuterium-labeled protein and efficiently utilize D2O and occasionally the deuterated substrate, fed-batch processes are routinely used to maximize biomass production without compromising cell viability. A step-by-step procedure will be described along with a case study of the production of deuterated green fluorescent protein. Limitations of the process will also be discussed.Research in recombinant protein expression in microorganism hosts spans half a century. The field has evolved from mostly trial-and-error approaches to more rational strategies, including careful design of the expression vectors and the coding sequence for the protein of interest. It is important to reflect on many aspects about vector construction, such as codon usage, integration site, coding sequence mutagenesis and many others. In this chapter, we overview methods and considerations to generate a suitable construct and anticipate possible experimental roadblocks.Fed-batch processes are commonly used in industry to obtain sufficient biomass and associated recombinant protein or plasmids. In research laboratories, it is more common to use batch cultures, as the setup of fed-batch processes can be challenging. This method outlines a robust and reliable means to generate Escherichia coli biomass in a minimum amount of fermentation time using a standardized fed-batch process. Final cell densities can reach over 50g dry cell weight per liter (gdcw/L) depending on the strain. This method uses a predefined exponential feeding strategy and conservative induction protocol to achieve these targets without multiple trial and error studies. If desired, productivity can be optimized by balancing the induction time and feed rates. This method utilizes cost-efficient defined media, minimizes process control complexity, and potentially aids downstream purification.A protocol for increasing soluble protein expression by fusing the chaperone GroEL apical domain with a gene of interest is described herein. GroEL apical domain, the minichaperone that functions independently of GroES and ATP in protein folding, is cloned downstream of the lambda CII ribosome binding site in the parent pRE vector. The pRE vector has tightly controlled transcription suitable for expressing toxic proteins. The GroEL minichaperone is fused to a glycine-serine rich linker followed by the enterokinase protease recognition sequence. A number of genes that are recalcitrant to protein production in the parent pRE vector 5were cloned into the pREGroEL fusion vector and successfully expressed as fusion proteins in Escherichia coli.Expression of heterologous genes in Escherichia coli is a routine technology for recombinant protein production, but the predictable recovery of properly folded and uniformly bioactive material remains a challenge. Misfolded proteins typically accumulate as insoluble inclusion bodies, and a variety of strategies have been employed in efforts to increase the yield of soluble product. One technique is the overexpression of E. coli protein chaperones during recombinant protein induction, in an effort to increase the folding capacity of the bacterial host. We have developed an alternative approach, by supplementing the host protein folding machinery with chaperones from other species. Extremophiles have evolved under conditions (extremes of temperature, salinity, pressure, and/or pH) that make them attractive candidates for possessing chaperones with novel folding activities. The green fluorescent protein (GFP) of Aequorea victoria, which is predominantly insoluble under typical recombinant expression culture conditions, was employed as an in vivo indicator of protein folding activity for chaperone homologs from a variety of extremophiles. For a subset of the chaperones tested, co-expression with GFP promoted an increase in both fluorescence signal intensity as well as the amount of GFP recovered in the soluble protein fraction. Several archaeal chaperones were also found to be able to refold soluble Lyt_Orn C40 peptidase from inclusion bodies in vitro. In particular, Pf Cpn(MA), a mutant chaperonin which exhibited significant refolding activity, is also shown to deconstruct the morphology and structure of inclusion bodies (Kurouski et al., 2012). Hence, the simple and rapid GFP assay provides a tool to screen for extremophilic chaperones that exhibit folding activity under E. coli growth conditions, and suggests that increasing the repertoire of heterologous chaperones might provide a partial but general solution to the problem of recombinant protein insolubility.Antibodies are globally important macromolecules, used for research, diagnostics, and as therapeutics. The common mammalian antibody immunoglobulin G (IgG) is a complex glycosylated macromolecule, composed of two heavy chains and two light chains held together by multiple disulfide bonds. For this reason, IgG and related antibody fragments are usually produced through secretion from mammalian cell lines, such as Chinese Hamster Ovary cells. However, there is growing interest in production of antibodies in prokaryotic systems due to the potential for rapid and cheap production in a highly genetically manipulable system. Research on oxidative protein folding in prokaryotes has enabled engineering of Escherichia coli strains capable of producing IgG and other disulfide bonded proteins in the cytoplasm, known as SHuffle. In this protocol, we provide a review of research on prokaryotic antibody production, guidelines on cloning of antibody expression constructs, conditions for an initial expression and purification experiment, and parameters which may be optimized for increased purification yields.