Sufficient Amounts Of Protein Research Articles

Purification of the alpha and beta subunits of (Na,K)-ATPase by continuous elution electrophoresis.

Covalent structural information on membrane proteins is not easily acquired since it is difficult to obtain pure membrane proteins in sufficient quantities. We have therefore examined the Bio-Rad 491 prep cell continuous elution electrophoresis apparatus as a method for providing the quantities of purified alpha and beta subunits from (Na,K)-ATPase required for these studies. Twenty-four milligrams of crude (Na,K)-ATPase preparation was applied to the prep cell which consisted of a 7% Laemmli separating gel 4.5 cm in length. The prep cell was run under constant power and continuous cooling conditions. Those fractions containing the beta subunit were combined and further purified by wheat germ agglutinin affinity chromatography. Fractions containing the alpha subunit were combined and did not require further purification. The identity and the degree of purity of the proteins obtained using this approach was assessed utilizing SDS-PAGE, amino acid analysis and N-terminal sequencing. This simple and fast method provides approximately 1.8 milligrams of each purified subunit from 24 milligrams of relatively crude microsomes. Recovery of the alpha and beta subunits from the crude (Na,K)-ATPase preparation was estimated to be 28% and 81%, respectively.

Nephila Clavipes Dragline Silk: Approaches to a Recombinantly Produced Silk Protein

Spider silks exhibit an unusual combination of strength and toughness that distinguishes them from other natural and synthetic fibers. Silk proteins perform a key natural function as structural fibers, to absorb impact energy from flying insects without breaking. They dissipate energy over a broad area and balance stiffness, strength and extensibility (1,2). In addition to their unusual mechanical properties and visual lustre, silks also exhibit interesting interference patterns within the electromagnetic spectrum (3), unusual viscometric patterns related to processing (4), and piezoelectric properties (3,5,6). These properties suggest they would be good candidates for high performance fiber and composite applications. However, the spider is not capable of producing sufficient quantities of proteins to enable thorough evaluation of their potential. Consequently, we are pursuing recombinant DNA techniques to clone and express adequate quantities of recombinant spider silk for these studies.

The impact of biotechnology and molecular biology on the pharmaceutical industry

Synopsis:Biotechnology had its initial impact on the pharmaceutical industry well before the perceived time. The use of fermentation technology to produce antibiotics was a cornerstone for the development of the industry. This event was both before cloning (BC) and before DNA (rather than after DNA – AD). Even now the antibiotic market, which is worth over 10 billion U.S. dollars a year, is the most valuable segment of the total market, (c.200 billion dollars per year). Nevertheless the impact of biotechnology in drug discovery was until recently perceived solely to be the use of recombinant DNA techniques to produce therapeutic proteins and modified versions of them by protein engineering.There are several other places where genetic engineering is influencing drug discovery. The expression of recombinant proteins in surrogate systems (e.g. in E. coli, yeast or via baculovirus infection or in mammalian cells) provides materials for structure determination (e.g. HIV protease) and structure/function studies (e.g. various receptors). Recombinant DNA techniques are influencing assay technology by allowing access to proteins in sufficient quantity for high throughput screening.In addition, screening organisms can be constructed where a particular protein function can be measured in a microorganism by complementation or via reporter gene expression.Transgenic animals also illustrate the power of the technology for drug discovery. Not only will transgenic rats and mice be used as models of disease but also for efficacy and toxicological profiling. What is learned in transgenic rodents may well set the scene for somatic cell gene therapy in humans.

Large scale expression and purification of recombinant HIV-1 proteinase from Escherichia coli

The availability of target proteins in sufficient quantity is a limiting factor in crystallographic studies and therefore in rational drug design. Even after optimisation, expression of recombinant proteins may be low and the only way to produce enough protein is by large scale cell growth/purification. HIV-1 proteinase in Escherichia coli, which due to its toxicity is expressed as a soluble protein only at around 0.1% of total protein, is a paradigm for this. In this paper a detailed process for large scale expression and purification of HIV-1 proteinase which delivers material of suitable quantity (30 mg from 500 g of wet weight of cells) and quality for crystallographic studies is described.

Purification of a membrane glycoprotein with an inositol-containing phospholipid anchor from Dictyostelium discoideum

Large-scale purification of a Dictyostelium discoideum cell surface glycoprotein, which is anchored in the membrane via a glycosylphosphatidylinositol (GPI) moiety, is described. The purification protocol involved four steps: separation of crude cell membranes by low-speed centrifugation, delipidization of these membranes using acetone, extraction of the membrane proteins using the detergent Octyl β-D-thioglucopyranoside (OTP), and purification of a specific membrane protein by monoclonal antibody immunoaffinity chromatography. The protein purified, PsA (prespore-specific antigen), is a developmentally regulated membrane glycoprotein found on a subset of cells from the cellular slime mould, D. discoideum. The protocol provides an efficient, economical, and technically simple way to purify GPI proteins in sufficient quantities for structural and functional studies. PsA was recovered at a yield of about 60%; with a purity of 97%, the extraction of 1 × 10 10 cells (1.1 g dry weight) yielded about 0.5 mg PsA glycoprotein. Techniques are described for growing kilogram quantities of D. discoideum cells in stainless steel trays at little cost. D. discoideum has considerable potential as a novel expression system for the production of foreign membrane-associated proteins. The purification strategy provides a means of purifying other GPI proteins, including those produced by protein engineering techniques.

31] Phycobiliproteins

This chapter focuses on Phycobiliproteins. In intact cyanobacterial cells and red algal chloroplasts, the phycobiliproteins are components of a complex assemblage, the phycobilisome. The observation that upon cell breakage the products of phycobilisome dissociation are released in water-soluble form gave rise to the unfortunate impression that the phycobiliproteins represented a fairly simple family of well-behaved macromolecules. Appraisal of the true complexity of the situation required an understanding of the molecular structure of phycobilisomes. During purification procedures, certain phycobilisome polypeptide components are lost through retention on chromatographic supports, or, in some cases, destroyed by proteolysis. Some phycobiliproteins, such as allophycocyanin B, are products of subunit scrambling during purification procedures, while others, such as the β18.3 polypeptide of the phycobilisome core, which is present in only two copies per phycobilisome, may copurify undetected with one of the major phycobiliproteins such as allophycocyanin. A polypeptide pattern obtained by SDS-polyacrylamide gel electrophoresis is an absolutely essential element in the characterization of a purified phycobiliprotein preparation. A sufficient amount of protein should be applied to the gel to ensure that components present in small amounts are detected.

Identification of a long form of cystatin from human saliva by rapid microbore HPLC mapping

Microbore HPLC methodology permits rapid and sensitive mapping of human saliva proteins. Saliva is sampled and processed in less than one hour, greatly reducing the likelihood of artifactual protein degradation. As little as 50 microliters of saliva yields proteins in sufficient quantities and purity to obtain amino terminal sequences directly. By this route we have discovered a 14 kDa protein extremely homologous to Cystatin S, but amino-terminally extended by eight amino acids.

Expression of the herpes simplex virus type-2 major DNA-binding protein (ICP8) gene in COS-1 cells

By the use of an SV40 origin of replication plasmid vector and the COS-1 cell system, expression of the gene encoding the herpes simplex virus type-2 (HSV-2) major DNA binding protein (ICP8) has been achieved. The HSV-2 4.5 kb BgIII 0 DNA fragment containing the ICP8 coding region was inserted into the plasmid vector pSVOd containing the SV40 origin of replication. Transfection of COS-1 cells by the resultant recombinant plasmid (pSVOd20), and subsequent multiplication of this plasmid, led to the production of the HSV-2 major DNA binding protein in sufficient quantities to allow its detection with either monoclonal or polyclonal antibodies as well as sera taken from HSV-2 patients.

Identification of monoclonal antibodies against the trypomastigote stage of Trypanosoma cruzi by use of iminobiotinylated surface polypeptides

The surface polypeptides of epimastigotes and tissue culture-derived trypomastigotes of Trypanosoma cruzi have been isolated free of most cytosolic components by use of the 2-iminobiotin-avidin interaction. Polypeptides of the trypomastigote stage obtained by this technique are recognized by serum antibodies from Chagasic patients and T. cruzi-infected mice. These polypeptides have been used as the detecting antigen for the identification of hybridoma cells producing monoclonal antibodies against the surface proteins of the trypomastigote stage of T. cruzi. These experiments document a practical approach for obtaining T. cruzi surface proteins in sufficient quantity and purity for use in immunological studies.

A plasma protein fractionation procedure for use in studies of protein metabolism.

A two-step method for the separation of five different plasma proteins on a preparative scale, which is capable of being extended to allow the separation of other plasma proteins, is described. The proteins separated were fibrinogen, two alpha(1)-glycoproteins, albumin and transferrin. The alpha(1)-glycoproteins were characterized in terms of electrophoretic mobility, ultracentrifugal and immunological characteristics. By using this method, it was shown that a single sample of plasma could be fractionated to yield purified proteins in sufficient quantity to simultaneously measure the synthesis of the two alpha(1)-glycoproteins, albumin and transferrin in the rat with McFarlane's technique (McFarlane, 1963; Reeve et al., 1963; McFarlane et al., 1965).

STUDIES ON THE METABOLISM OF ALPHA-LIPOIC ACID IN CHILDHOOD. 3. EFFECT OF DIETARY COMPOSITION AND ANBOLIC STEROID.

The effect of dietary composition and anabolic steroid hormone on the lipoic acid metabolism in weaning and mature rats were observed and the following findings were obtained.1. The urinary excretion for 24 hours, and the levels in the serum, liver and kidney of lipoic acid in mature rats were, on an average, 77mμg, 17.5mμg/ml, 2880mμg/g and 2700mμg/g, respectively and the nitrogen contents of the liver and kidney were 29.0 and 31.6mg/g respectively.2. The urinary excretion and the serum level of lipoic acid in the low protein diet group were slightly lower than those in the standard diet group. In such animals, the lipoic acid content in the liver was remarkably decreased in correlation with the decline of nitrogen level.3. The influence of a low protein diet was stronger in the weaning rats than in the mature ones. The lipoic acid level in the liver was decreased to 50% of the normal level.4. The effect of amino acid deficiency of the diet on the content of lipoic acid in the liver was examined and the influence of tryptophan deficiency was the strongest, followed by that of lysine deficiency.5. The effect of the anabolic steroid hormone on the weight gain, serum protein concentration, urinary excretion of lipoic acid, serum lipoic acid level, weight of the liver, kidney, or testicle, and nitrogen and lipoic acid contents of these tissues under several different conditions of the diet in rats were investigated.The anabolic effect on nitrogen metabolism and the influence on the lipoic acid metabolism by this type of hormone were most remarkable when the rats were fed at first the low protein diet and then that with a sufficient quantity of protein. On the contrary, the catabolic effect of 19-norandrosterone phenylpropionate was observed in the rats fed a sufficient or low protein diet.

Ascorbic Acid Synthesis is Rats on Various Diets

Summary. I. The ascorbic acid excretion in the urine has been studied in rats on varying diets. A normal excretion could be maintained, when the diet contained sufficient quantities of protein (casein) and riboflavin. Addition of anuran, niacin, alderman and antithetic acid had no effect. This also held good when the ascorbic-acid excretion was increased by the administration of an aromatic compound, e. g. azotoluene. The experiences from these experiments indicate that the casein is necessary for the synthesis of ascorbic acid and does not merely have a stimulating effect, as e. g. azotoluene; an increased ascorbic-acid excretion after administration of azotoluene was only obtained when the diet contained sufficient quantities of casein (and riboflavine). In agreement with the excretion experiments rats, with a deficit of riboflavine show a decreased ascorbic-acid content in the liver. II. The variations observed in ascorbic-acid content in the liver and in the ascorbic-acid excretion should correspond to the variations in the synthesis of ascorbic acid and not only to a varying metabolism. III. If rats with a deficit of riboflavin receive ascorbic acid, the period of survival is increased. In experiments with administration of sulfanilamide to rats ca. 100 % of the quantities administered were excreted at a riboflavin deficit; after administration of riboflavin or ascorbic acid only about 50 %. In experiments with administration of azotoluene only small quantities of the oxidized decomposition product m-aminobenzoic acid were excreted in riboflavine deficit; after supply of riboflavine (in a sufficient addition of casein) or ascorbic acid, considerably increased quantities were excreted. It is considered that the ascorbic acid is of importance for oxidizing detoxication processes in the organism of the rat. Riboflavine, by its effect on the ascorbic-acid production, can have a similar influence.

Ein neuentdeckter Wirkstoff (Vitamin-T-Komplex)

The recently discovered vitamin T complex can be extracted from different kinds of yeast and ascomycets, but also from insects that feed on such micro-organisms. In case of a sufficient amount of protein in the food, vitamin T causes an “acceleration of development”; thus it is possible that insects may attain new and varied proportions of their bodies, such as would never occur in nature (“Great modification”). With vertebrates, the assimilation of protein is increased by vitamin T. The result is an enlargement of the body and an increase in weight of from 10 to 20%, in spite of the same or even a smaller amount of nourishment. Also with human beings an increase in weight of 3 to 7 pounds was observed within 20 days, though the small rations were not increased. Besides, vitamin T has a favourable influence on healing wounds. The importance of vitamin T for agriculture and medicine is obvious.