Leif Bülow

Metal-binding proteins and peptides in bioremediation and phytoremediation of heavy metals

The expression of metal-binding proteins or peptides in microorganisms and plants in order to enhance heavy metal accumulation and/or tolerance has great potential. Several different peptides and proteins have been explored. This review focuses on cadmium (Cd) because of the significant importance of this metal and because of its global presence in many food materials.

Improving stress tolerance in plants by gene transfer

Plant productivity is greatly influenced by environmental stresses, such as freezing, drought, salinity and flooding. One of the ways in which tolerance to these factors can be achieved is by the transfer of genes encoding protective proteins or enzymes from other organisms. Key approaches currently being examined are engineered alterations in the amounts of osmolytes and osmoprotectants, saturation levels of membrane fatty acids, and rate of scavenging of reactive oxygen intermediates.

Chromatography of microbial cells using continuous supermacroporous affinity and ion-exchange columns

Continuous supermacroporous chromatographic columns with anion-exchange ligands [2-(dimethylamino)ethyl group] and immobilized metal affinity (IMA) ligands (Cu2+-loaded iminodiacetic acid) have been developed allowing binding of Escherichia coli cells and the elution of bound cells with high recoveries. These poly(acrylamide)-based continuous supermacroporous columns have been produced by radical co-polymerization of monomers in aqueous solution frozen inside a column (cryo-polymerization). After thawing, the column contains a continuous matrix (so-called cryogel) with interconnected pores of 10-100 microm in size. The large pore size of the matrix makes it possible for E. coli cells to pass unhindered through a plain column containing no ligands. E. coli cells bound to an ion-exchange column at low ionic strength were eluted with 70-80% recovery at NaCl concentrations of 0.35-0.40 M, while cells bound to an IMA-column were eluted with around 80% recovery using either 10 mM imidazole or 20 mM EDTA solutions, respectively. The cells maintain their viability after the binding/elution procedure. These preliminary results indicate that microbial cells can be handled in a chromatographic mode using supermacroporous continuous columns. These columns are easy to manufacture from cheap and readily available starting materials, which make the columns suitable for single-time use.

Functional expression of five Arabidopsis fatty acyl-CoA reductase genes in Escherichia coli.

Very long chain primary alcohols are significant components in cuticle waxes of plants. Fatty acyl-CoA reductases (FARs) catalyze the formation of a fatty alcohol from an acyl-CoA. The Arabidopsis (Arabidopsis thaliana) genome contains eight genes homologous to FAR genes from jojoba (Simmondsia chinensis), silk moth, wheat and mouse. Expression of six Arabidopsis FAR homologs in Escherichia coli resulted in production of alcohols from endogenous E. coli fatty acids by five of these genes, confirming that they encode for FAR enzymes. Only a truncated splicing version of the sixth gene was found, and this gene yielded a protein with no FAR activity. The five functional FAR enzymes yielded distinctly different compositions of fatty alcohols when expressed in E. coli, indicating that the different enzymes may be involved in the production of different types of alcohols in plant cells.

Multienzyme systems obtained by gene fusion

The preparation of artificial bi- and polyfunctional enzymes by gene fusion appears to have great potential in enzyme technology. Their purification is facilitated compared with that of their naturally occurring counterparts and they exhibit favourable enzyme kinetics. Applications can be found in biochemical analysis, enzyme process technology and metabolic engineering.

Tyrosine Residues as Redox Cofactors in Human Hemoglobin IMPLICATIONS FOR ENGINEERING NONTOXIC BLOOD SUBSTITUTES

Respiratory proteins such as myoglobin and hemoglobin can, under oxidative conditions, form ferryl heme iron and protein-based free radicals. Ferryl myoglobin can safely be returned to the ferric oxidation state by electron donation from exogenous reductants via a mechanism that involves two distinct pathways. In addition to direct transfer between the electron donor and ferryl heme edge, there is a second pathway that involves “through-protein” electron transfer via a tyrosine residue (tyrosine 103, sperm whale myoglobin). Here we show that the heterogeneous subunits of human hemoglobin, the α and β chains, display significantly different kinetics for ferryl reduction by exogenous reductants. By using selected hemoglobin mutants, we show that the α chain possesses two electron transfer pathways, similar to myoglobin. Furthermore, tyrosine 42 is shown to be a critical component of the high affinity, through-protein electron transfer pathway. We also show that the β chain of hemoglobin, lacking the homologous tyrosine, does not possess this through-protein electron transfer pathway. However, such a pathway can be engineered into the protein by mutation of a specific phenylalanine residue to a tyrosine. High affinity through-protein electron transfer pathways, whether native or engineered, enhance the kinetics of ferryl removal by reductants, particularly at low reductant concentrations. Ferryl iron has been suggested to be a major cause of the oxidative toxicity of hemoglobin-based blood substitutes. Engineering hemoglobin with enhanced rates of ferryl removal, as we show here, is therefore likely to result in molecules better suited for in vivo oxygen delivery.

Formation of proinsulin by immobilized Bacillus subtilis.

There has been an increasing interest in the use of immobilized cells for the production of pharmaceuticals as well as for products such as high fructose syrup or ethanol1. Some of these compounds are now produced on an industrial scale2 whereby the cells are used in a resting or growing state or in a nonviable form as natural carriers of the enzyme(s) involved in the synthesis. The advantages of immobilized cell technology should also apply to microorganisms modified by recombinant DNA techniques to produce a variety of eukaryotic proteins such as hormones. We describe here the properties of immobilized Bacillus subtilis cells carrying plasmids encoding rat proinsulin. Cell proliferation normally coupled to DNA replication is undesirable in immobilized cell systems as ‘clogging’ of the system occurs due to cells growing outside the beads. Therefore, different ways were investigated to inhibit cell division while allowing continued protein synthesis. We found that the addition of certain antibiotics in the growth medium, such as novobiocin which inhibits DNA replication3, fulfills these requirements, allowing proinsulin synthesis and excretion to take place over a period of several days.

Pathological Conditions Involving Extracellular Hemoglobin: Molecular Mechanisms, Clinical Significance, and Novel Therapeutic Opportunities for alpha(1)-Microglobulin

Hemoglobin (Hb) is the major oxygen (O(2))-carrying system of the blood but has many potentially dangerous side effects due to oxidation and reduction reactions of the heme-bound iron and O(2). Extracellular Hb, resulting from hemolysis or exogenous infusion, is shown to be an important pathogenic factor in a growing number of diseases. This review briefly outlines the oxidative/reductive toxic reactions of Hb and its metabolites. It also describes physiological protection mechanisms that have evolved against extracellular Hb, with a focus on the most recently discovered: the heme- and radical-binding protein α(1)-microglobulin (A1M). This protein is found in all vertebrates, including man, and operates by rapidly clearing cytosols and extravascular fluids of heme groups and free radicals released from Hb. Five groups of pathological conditions with high concentrations of extracellular Hb are described: hemolytic anemias and transfusion reactions, the pregnancy complication pre-eclampsia, cerebral intraventricular hemorrhage of premature infants, chronic inflammatory leg ulcers, and infusion of Hb-based O(2) carriers as blood substitutes. Finally, possible treatments of these conditions are discussed, giving a special attention to the described protective effects of A1M.

Haptoglobin Binding Stabilizes Hemoglobin Ferryl Iron and the Globin Radical on Tyrosine β145

AIM Hemoglobin (Hb) becomes toxic when released from the erythrocyte. The acute phase protein haptoglobin (Hp) binds avidly to Hb and decreases oxidative damage to Hb itself and to the surrounding proteins and lipids. However, the molecular mechanism underpinning Hp protection is to date unclear. The aim of this study was to use electron paramagnetic resonance (EPR) spectroscopy, stopped flow optical spectrophotometry, and site-directed mutagenesis to explore the mechanism and specifically the role of specific tyrosine residues in this protection. RESULTS Following peroxide challenge Hb produces reactive oxidative intermediates in the form of ferryl heme and globin free radicals. Hp binding increases the steady state level of ferryl formation during Hb-catalyzed lipid peroxidation, while at the same time dramatically inhibiting the overall reaction rate. This enhanced ferryl stability is also seen in the absence of lipids and in the presence of external reductants. Hp binding is not accompanied by a decrease in the pK of ferryl protonation; the protonated ferryl species still forms, but is intrinsically less reactive. Ferryl stabilization is accompanied by a significant increase in the concentration of the peroxide-induced tyrosine free radical. EPR spectral parameters and mutagenesis studies suggest that this radical is located on tyrosine 145, the penultimate C-terminal amino acid on the beta Hb subunit. INNOVATION Hp binding decreases both the ferryl iron and free radical reactivity of Hb. CONCLUSION Hp protects against Hb-induced damage in the vasculature, not by preventing the primary reactivity of heme oxidants, but by rendering the resultant protein products less damaging.

Enzyme purification by genetically attached polycysteine and polyphenylalanine affinity tails.

Two novel affinity tails, polycysteine and polyphenylalanine, have been genetically attached to galactokinase (EC 2.7.1.6) and beta-galactosidase (EC 3.2.1.23) in order to facilitate their purification. A chemically synthesized DNA linker encoding four cysteine residues was thus fused in frame with the galactokinase gene. The gene product, cysteine galactokinase, was significantly retarded on a column of thiopropyl-Sepharose. Using pulse elution, cysteine galactokinase was eluted at 10 mM DTT. Under the condition used, native galactokinase did not bind to thiopropyl-Sepharose. Homopolymer tailing was employed to prepare a phenylalanine-modified beta-galactosidase. One of the obtained genetic transformants coding for a beta-galactosidase carrying 11 phenylalanine residues at the N-terminus of the enzyme was isolated. With the aid of hydrophobic interaction chromatography the modified enzyme could be purified to homogeneity on fast protein liquid chromatography using a phenyl-Superose column.