Optimization of elastin‐like polypeptide fusions for expression and purification of recombinant proteins in plants

The demand for recombinant proteins for medical and industrial use is expanding rapidly and plants are now recognized as an efficient, inexpensive means of production. Although the accumulation of recombinant proteins in transgenic plants can be low, we have previously demonstrated that fusions with an elastin‐like polypeptide (ELP) tag can significantly enhance the production yield of a range of different recombinant proteins in plant leaves. ELPs are biopolymers with a repeating pentapeptide sequence (VGVPG)_n that are valuable for bioseparation, acting as thermally responsive tags for the non‐chromatographic purification of recombinant proteins. To determine the optimal ELP size for the accumulation of recombinant proteins and their subsequent purification, various ELP tags were fused to green fluorescent protein, interleukin‐10, erythropoietin and a single chain antibody fragment and then transiently expressed in tobacco leaves. Our results indicated that ELP tags with 30 pentapeptide repeats provided the best compromise between the positive effects of small ELP tags (n = 5–40) on recombinant protein accumulation and the beneficial effects of larger ELP tags (n = 80–160) on recombinant protein recovery during inverse transition cycling (ITC) purification. In addition, the C‐terminal orientation of ELP fusion tags produced higher levels of target proteins, relative to N‐terminal ELP fusions. Importantly, the ELP tags had no adverse effect on the receptor binding affinity of erythropoietin, demonstrating the inert nature of these tags. The use of ELP fusion tags provides an approach for enhancing the production of recombinant proteins in plants, while simultaneously assisting in their purification. Biotechnol. Bioeng. 2009;103: 562–573. © 2009 Wiley Periodicals, Inc.     

Gender‐ and season‐dependent relationships between testosterone, oestradiol and immune functions in wild roach

Plasma testosterone and 17β‐oestradiol concentrations, differential leukocyte counts and proportion of dead Rhipidocotyle campanula gill parasites (parasite resistance) were determined five times during a year in two populations of roach Rutilus rutilus and analysed for seasonal and gender differences. In addition to the above immune variables, plasma Immunoglobulin M (IgM) concentration, chemiluminescence and migration differential of head kidney phagocytes, size of the spleen, haematocrit and total leukocyte count were correlated with sex hormones for each population, sampling time and sex separately, using condition factor as a partial correlate. There were no clear gender differences in the determined immune variables. Both testosterone and oestradiol concentrations were lowest after the spawning in June. Oestradiol was higher in females than in males, but testosterone was present in equal concentrations in both sexes. Statistically significant correlations between sex hormones and immune variables were rare and mainly without any patterns with respect to population, sex or sampling date. The meta‐analysis on individual correlations, however, revealed a positive relationship of plasma testosterone concentration to chemiluminescence of head kidney phagocytes, plasma IgM concentration and the proportion of dead R. campanula to all R. campanula on the gills. In the meta‐analysis, the concentration of oestradiol was not found to correlate with any of the studied immune variables.     

Transformable mutants of a biopesticide strainStreptomyces griseoviridis K61

Summary The aim of this work was to isolate transformable mutants ofStreptomyces griseoviridis K61 without affecting the secondary metabolism of this strain.S. griseoviridis K61 produces an antifungal aromatic heptaene polyene antibiotic, and is used as a biological control agent. In protoplast transformation experiments using plasmid pIJ702 DNA, the few spontaneous transformants were phenotypically bald and their secondary metabolism was pleiotropically affected. By mutagenizing K61 withN-methyl-N-nitro-N-nitrosoguanidine (MNNG) a highly transformable variant K61-42 was obtained. Protoplasts ofS. griseoviridis K61-42 could be transformed by several model plasmids producing 10⁴–10⁵ transformants/g plasmid DNA. The polyene synthesis of K61-42 was normal, making this strain a useful tool in genetic studies on the mechanism of biopesticide action.     

Purification of intrinsic factor receptor from pig ileum using as affinity medium human intrinsic factor covalently bound to Sepharose

Intrinsic factor receptor was purified from hog ileum using human intrinsic factor covalently bound to Sepharose. A yield of 49.6% and a specific activity of about 2500 pmol/mg protein were achieved. The purified receptor was very unstable: 24 h of storage or addition of sodium phosphate precipitated it. The association constant of the receptor for the cyano[57Co]cobalamin-intrinsic factor complex was estimated to be 2.1 nM-1. In native polyacrylamide gel electrophoresis it resolved in two 256 and 320 kDa bands; beta-mercaptoethanol treatment cleared it into four bands corresponding to molecular masses of 107, 81.8, 63.5 and 53.2 kDa. An additional 39.3 kDa band was considered to be an artefact due to the presence of Triton X-114. Isoelectric focusing polyacrylamide gel electrophoresis resolved the receptor into two isoproteins isoelectric at pH 4.7 and 5.1. A similar result was obtained in column electrofocusing with the 125I-iodinated receptor. The 125I-labelled receptor did not crossreact with rabbit anti-human intrinsic factor antiserum. The electrophoretic properties of the receptor purified with intrinsic factor covalently bound to Sepharose were compared to those of the receptor purified by the use of the classical cobalamin-affinity medium. It was concluded that a disassembled receptor was produced using the classical method.     

Extracellular adhesive molecules in neurite growth

This review deals with two topics: (1) the effects of fibronectin and laminin on neurite growth and the molecular mechanisms of these effects, and (2) isolation and properties of the adhesive molecule p30. This novel molecule is an abundant heparin-binding protein in perinatal rat brain, and is suggested to have a role in neuronal growth.     

Reduction of Nitrate via a Dicarboxylate Shuttle in a Reconstituted System of Supernatant and Mitochondria from Spinach Leaves

Substantial rates of nitrate reduction could be achieved with a reconstituted system from spinach leaves containing supernatant, mitochondria, NAD⁺, oxaloacetate (OAA), and an oxidizable substrate. Appropriate substrates were glycine, pyruvate, citrate, isocitrate, fumarate, or glutamate. The reduction of NO₃⁻ with any of the substrates could be inhibited by n-butyl malonate, showing that the transfer of reducing power from the mitochondria to the supernatant involved the malate exchange carrier. The addition of ADP to the reconstituted system decreased NO₃⁻ reduction and this decrease could be reversed by the addition of rotenone or antimycin A. The operation of the OAA/malate shuttle was achieved most quickly in the system when low concentrations (≤0.1 millimolar) of OAA were added. A corresponding increase in the lag time for the operation of the OAA/malate shuttle was observed when the OAA concentration was increased. Concentrations for half-maximal activity of OAA, glycine, NAD⁺, and NO₃⁻ in the reconstituted system were 42 micromolar, 0.5 millimolar, 0.25 millimolar, and 26 micromolar, respectively. The transfer of reducing power from the mitochondria to the soluble phase via the OAA/malate shuttle can not only provide NADH for cytoplasmic reduction but can also sustain oxidation of tricarboxylic cycle acids and the generation of α-ketoglutarate independently of the respiratory electron transport chain.     

Environment-dependent mechanism of dehalogenation by Rhodococcus chlorophenolicus PCP-1

Cells and cell-free preparations of a soil-bioremediating organism, Rhodococcus chlorophenolicus PCP-1, dehalogenated polychlorophenols both under aerobic and anaerobic conditions. Under aerobic conditions molecular O₂ served as the source of oxygen for the dechlorinating para-hydroxylation reaction. Chlorophenols were dehalogenated and para-hydroxylated also under anaerobic conditions by a cyt P-450 enzyme. Water was used anaerobically as an oxygen source but the reaction required the presence of sulphite ions or iodosobenzene. When the dehalogenating enzyme was given a choice between molecular O₂ and water in the presence of sulphite ions or iodosobenzene, the oxygen was preferably derived from water.Dedicated to the honour of Prof. Dr. Norberto Palleroni for the occasion of his 70th birthday Correspondence to: J. S. Uotila     

Kinetic model for the action of the inorganic pyrophosphatase from Streptococcus faecalis

Kinetic studies of the less active form of Streptococcus faecalis inorganic pyrophosphatase (EC 3.6.1.1), together with computational analysis, indicated that cooperativity in ligand binding contributes in a significant way to the behavior of this enzyme. The simplest model applicable to our data was a Monod-Wyman-Changeux-type, allosteric model, in which the enzyme is proposed to exist in two states, referred to as R and T states, respectively. In the absence of ligands, 94% of the enzyme was in the T state. MgPPi2- was the only substrate for the enzyme in the R form. This substrate was bound equally well by both enzyme forms, but it was hydrolyzed 5 times more efficiently by the R form than it was by the T form. Mg2PPi was bound exclusively to the T state of the enzyme, and it was hydrolyzed 25% as rapidly as MgPPi2- by the T form. Mg2PPi inhibited the hydrolysis of the more efficient substrate, MgPPi2-, by competing with MgPPi2- for the enzyme in the T form and by shifting the R----T equilibrium in favor of the T form. Mg2+ stabilized the R state, thus activating the hydrolysis of MgPPi2- and inhibiting that of Mg2PPi.     

Induction of delayed-type hypersensitivity with proteins made insoluble by polymerization

Protein antigens, made particulate by polymerization with ethyl chloroformate, were incorporated in Freund's complete adjuvant and used for footpad immunization of rats and guinea pigs. A comparison was made with animals similarly immunized with the native, soluble protein. Two to three weeks after immunization of rats with polymerized bovine serum albumin (Pol-BSA) and up to 8 weeks after immunization of guinea pigs with polymerized diphtheria toxoid, in vivo and in vitro evidence of delayed-type hypersensitivity (DTH) was found without measurable serum antibodies. Ten times more polymerized than soluble BSA was needed to induce comparable levels of DTH. This was not, however, true in the case of serum antibodies, since soluble BSA induced higher titers than the 1000 times larger amount of Pol-BSA. In addition, the titers in polymer-immunized rats were consistently low or under detectable level when followed up to 5 months after priming. These findings encourage the belief that insolubilization of antigens by polymerization guides the immune response toward cell-mediated immunity, whereas antibody formation becomes weaker. However, boosting of polymer-primed animals with soluble antigen resulted in the production of high levels of antibody.