A new method for obtaining human transferrin C-lobe in the native conformation: preparation and properties

Eukaryotic transferrins comprise a class of bilobal iron-binding proteins in which each lobe carries a single binding site. Although expression of full-length transferrins and their N-terminal lobes, in wild-type and mutated forms, has been successfully accomplished by several laboratories, expression of C-lobes has been much less satisfactory. A possible explanation of the difficulty is that proper folding of the C-lobe, with its 11 disulfide bonds, depends on prior synthesis and proper folding of the N-lobe. We have therefore developed a new strategy, introducing a specific factor Xa cleavage site in the interlobe-connecting strand to permit separation of the lobes after expression of the full-length protein. The resulting protein was expressed in satisfactory yield, >20 mg/L, and could be easily and completely cleaved to yield two distinguishable fragments representing N- and C-lobes, respectively. Retaining the glycosylation sites, found only in the C-lobe, made it possible to separate the fragments from each other by ConA affinity chromatography. The isolated C-lobe so obtained displayed spectroscopic and kinetic features of the C-lobe in native transferrin and was competent as an iron donor for K562 cells to which it bound in saturable fashion inhibitable by native diferric transferrin. Since the N-lobe by itself will neither bind nor donate iron to cells, the primary receptor-recognition site of transferrin resides in its C-lobe.     

Expression, purification, and characterization of recombinant nonglycosylated human serum transferrin containing a C-terminal hexahistidine tag

Attachment of a hexa-His tag is a common strategy in recombinant protein production. The use of such a tag greatly simplifies the purification of the protein from the complex mixture of other proteins in the media or cell extract. We describe the production of two recombinant nonglycosylated human serum transferrins (hTF-NG), containing a factor Xa cleavage site and a hexa-His tag at their carboxyl-terminal ends. One of the constructs comprises the entire coding region for hTF (residues 1-679), while the other lacks the final three carboxyl-terminal amino acids. After insertion of the His-tagged hTFs into the pNUT vector, transfection into baby hamster kidney (BHK) cells, and selection with methotrexate, the secreted recombinant proteins were isolated from the tissue culture medium. Average maximum expression levels of the His-tagged hTFs were about 40 mg/L compared to an average maximum of 50 mg/L for hTF-NG. The first step of purification involved an anion exchange column. The second step utilized a Poros metal chelate column preloaded with copper from which the His-tagged sample was eluted with a linear imidazole gradient. The His-tagged hTFs were characterized and compared to both recombinant hTF-NG and glycosylated hTF from human serum. The identity of each of the His-tagged hTFs constructs was verified by electrospray mass spectroscopy. In summary, the His-tagged hTF constructs simplify the purification of these metal-binding proteins with minimal effects on many of their physical properties. The His-tagged hTFs share many features common to hTF, including reversible iron binding, reactivity with a monoclonal antibody, and presence as a monomer in solution.     

A detailed method for obtaining preparations of human sperm chromosomes

A detailed technique is described for obtaining preparations of the chromosome complements of human sperm by fertilization of hamster eggs and analysis of the male pronucleus. Some of the more difficult aspects and important steps are emphasized. Technical data from 17 consecutive experiments are presented to provide an estimate of the number of karyotypes which can be obtained in an experiment.     

Production of a functional dihydrofolate synthase with a cleavable poly-His tag in Saccharomyces cerevisiae

Polyhistidine-tagged dihydrofolate synthase (DHFS) has been produced in the yeast, Saccharomyces cerevisiae, using a Cu²⁺-inducible expression system. The tagged DHFS is functional in vivo and was purified using immobilised metal affinity chromatography. A linker of a minimal size allows efficient cleavage of the poly-His tag using thrombin. At least 10 mg of pure DHFS can be recovered per litre of culture.     

Preliminary crystallographic analyses of the N-terminal lobe of recombinant human serum transferrin.

The N-terminal lobe of recombinant human serum transferrin (residues 1 to 337) has been crystallized in a form suitable for high-resolution three-dimensional X-ray crystallographic analyses. Crystals are of the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions of a = 44.9 A, b = 57.0 A and c = 135.9 A, and diffract to beyond 2 A resolution. Further studies show that isomorphous crystals of specifically designed mutants of this protein can also be grown. Structural studies of both recombinant and mutant protein forms will provide a basis for understanding the mechanism by which human serum transferrin functions.     

Preparation and properties of a single-sited fragment from the C-terminal domain of human transferrin

A single-sited iron-binding fragment of human transferrin has been obtained by thermolysin cleavage of the protein, selectively loaded with iron in the C-terminal binding site, in a urea-containing buffer. The fragment contains carbohydrate, and hence derives from the C-terminal half of transferrin. Its metal-binding site accepts Fe3+ and Cu2+ with bicarbonate as accompanying anion, but only Fe3+ with oxalate as anion. EPR spectroscopic properties of the fragment are similar to those of the corresponding site in the intact protein. However, iron-binding by the fragment is weaker than by the C-terminal site of the intact protein, particularly at low pH, suggesting that overall as well as local protein conformation influences the metal-binding functions of the site.     

A method for obtaining DNA from compost

An effective cell lysis method for extraction of bacterial genomic DNA from compost was developed in this study. Enzymatic disruption method, physical–chemical combination method, and commercial kit method were used to extract DNA from compost samples and were compared by analyzing DNA yield and efficient cell lysis. The results showed that all the three methods can be used to extract high-quality DNA from compost, but the enzymatic method had better cell lysis efficiency and DNA yields than others without the use of special equipment and expensive spending. Comparison of different methods for lysing gram-positive bacteria Bacillus subtilis indicated that the enzymatic cell lysis is superior for destroying the gram-positive cell wall. Spin-bind DNA column was used for DNA purification, and the purity of the purified sample was checked by polymerase chain reaction to amplify a region of the 16S rRNA. Results indicated that the part of 16S rRNA were amplified from all the purified DNA samples, and all the amplification products could be digested by the restriction enzyme HhaI.     

A mechanical method for obtaining soil cores

Summary Apparatus is described which enables large numbers of soil cores to be collected from depths up to 70 cm in small plot areas with little disturbance of the soil within the corers.     

A rapid method for obtaining monomeric glutaraldehyde

A new method for the distillation of glutaraldehyde to obtain the monomeric form is presented. The monomer is obtained after only one distillation and it has a purification index (Pi) smaller than 0.20.     

A rapid method for obtaining monomeric glutaraldehyde

Summary A new method for the distillation of glutaradlehyde to obtain the monomeric form is presented. The monomer is obtained after only one distillation and it has a purification index (P_i) smaller than 0.20.     

A method for obtaining protein concentrates from microorganisms

In order to isolate proteins from microalgae, yeasts and bacteria, cell disintegration in a special ball-mill was performed. The degree of disintegration of the different microorganisms was compared. The dependence of disintegration on bead size and on the ratio between the volume of suspension and the volume of glass beads was also investigated. Nondisintegrated and disintegrated cells were extracted with sodium hydroxide and the amount of extractable nitrogen and the amount of nitrogen precipitable at pH 4.0 were determined. The dependence of yield on the sodium hydroxide concentration, extraction time, and temperature was studied. When extracting undisintegrated cells, very low yields were obtained and the nitrogen extracted was mostly nonproteinous. For disintegrated cells high yields were obtained. An optimum was found after extraction with 0.3–0.5% sodium hydroxide; at pH 11.0–11.5. The precipitate obtained represented 60–70% of the cell nitrogen. The nitrogen content of the precipitate was 12–14% of the dry weight.     

A preparative method for obtaining enucleated mammalian cells.

Analysis of the 2D gel electrophoretic pattern of ribosomal proteins from both the small and large subunit of rat liver were made at various times following partial hepatectomy. No changes were observed in the electrophoretic mobility of proteins from the 60S subunit during periods of 2 hr. to 72 hr. of liver regeneration. Changes were observed, however, in two proteins of the 40S subunit a short time after partial hepatectomy. Protein S₆ disappeared from its normal position and a new spot appeared as a more negative form as early as 2 hr. post regeneration. This modification persisted for at least 18 hr. At 72 hr., S₆ returned to its normal position. Protein S₂, on the other hand, underwent a different pattern of change during the early stages of liver regeneration. S₂ was observed to migrate as 2 spots at 2 hr. after partial hepatectomy and this pattern was preserved at the 4 hr. period. At 8 hr., the pattern was further modified to 2 spots which was distinct from the earlier change. This pattern was similar at 12 hr. At 18 hr. only the normal S₂ protein was observed. No further change in S₂ migration was observed at the 72 hr. period of liver regeneration.     

A method for obtaining a long-lived intestinal tissue culture

The interest in the studies of the intestines using the method of tissue organ culture has considerably grown in recent years. It can be explained by the great possibilities of obtaining unique data about the state of intestines in normal and pathological condition, e.g. malabsorption syndrome. The paper deals with the method modified by the authors to obtain long-living (24 hours) intestinal tissue organ culture. The investigations used bioptic sections which were obtained by jejunoscopy with spot biopsy of children suffering from intestinal malabsorption. The viability of the explants was proved by histological and histochemical tests. The promise held by the methods is emphasized.