A novel method for purification of plasma fibronectin

Plasma fibronectin was purified from a gelatin-affinity chromatography column by elution with glucose. This procedure was effective only if the gelatin was particulate when it was attached to the Sepharose 4B. Glucose could not elute fibronectin from the gelatin if the gelatin was melted before it was attached to the Sepharose 4B. This new purification technique has the advantage of using very mild conditions for the isolation of plasma fibronectin.     

A novel method for purification of prephenic acid.

Prephenic acid accumulated in culture filtrates of Neuro-spora crassa has been purified in 66% yield utilizing adsorption chromatography on Sephadex G-10 in the major purification steps.     

A novel purification method for multipotential skeletal stem cells

At least some cells within bone marrow stromal populations are multipotential (i.e., differentiate in vitro into osteoblasts, chondrocytes, and adipocytes) and thus designated skeletal stem cells (SSCs) or mesenchymal stem cells (MSCs) amongst other names. Recently, a subpopulation of stromal cells, notably osteoblasts or their progenitors, has been identified as a definitive regulatory component of the hematopoietic stem cell (HSC) niche. Thus, the development of methods for purifying not only SSCs but cells comprising the HSC niche is of interest. Here, we report a method for purifying a novel bone marrow‐derived population with a high frequency of osteoprogenitors and high expression levels of osteoblast differentiation markers (highly purified osteoprogenitors (HipOPs)) as well as markers of the bone niche for HSCs. In vivo transplantation experiments demonstrated that donor HipOPs differentiated into not only osteoblasts, osteocytes and cells around sinusoids but also hematopoietic cells. Thus, HipOPs represent a novel population for simultaneous reconstruction of bone and bone marrow microenvironments. J. Cell. Biochem. 108: 368–377, 2009. © 2009 Wiley‐Liss, Inc.     

A novel concept in the purification of human leukocyte interferon

A method for the purification of natural human interferon alpha (HuIFN Alpha) is described. It involves adsorption of interferon on silicic acid and its elution with hydrophobic electrolyte solution. Thereafter, elimination of possible viruses is achieved using nonionic detergent and ultrafiltration. Interferon recovered in the ultrafiltrate is further purified on Sephacryl S-200. Fractions corresponding to molecular weights ranging from approximately 10,000 to approximately 40,000 daltons are collected and directly applied on immobilized zinc affinity gel. IFN Alpha which is eluted with the non adsorbed fraction is subjected to buffer exchange, concentration, and sterilization. The resulting solution shows high specific activity (1 x 10(7) IU) with an apparently natural composition of interferon isospecies. The uniform buffer employed through the entire purification process makes it simple, fast and reproducible.     

Diol sulfonamides: A potent and novel class of inhibitors of human renin

The syntheses and pharmacological activity of a series of diol sulfonamides which function as inhibitors of human renin are described. The most potent compound in this series, compound 20 (SQ 33,800), is a subnanomolar inhibitor of human renin (IC₅₀ = 0.35 × 10⁻⁹ M).     

A novel laboratory method of isolation and purification of tobramycin]

A new laboratory method for isolation and purification of tobramycin by using extraction of a tobramycin derivative with benzaldehyde by methylene chloride, subsequent hydrolysis of azomethine and recrystallization of the formed tobramycin sulfate from solution of sulfuric acid in methanol was developed. The method allows to exclude the stage of chromatographic purification of tobramycin, to reduce the time of the process realization from 120-125 h to 15-20 h, to increase the yield of the target product from 37-40% to 60-65% without decreasing the product quality, to exclude a number of large-size and expensive equipment and to ensure high reproducibility of the technology.     

A novel method for simultaneous purification of albumin and immunoglobulin G

A novel method was developed to obtain both highly purified bovine serum albumin (BSA) and immunoglobulin G (IgG), at the same time, on a pilot scale. Heat-isopropyl alcohol was used to denature and precipitate the other plasma proteins, except for BSA and IgG; then, CM-Trisacryl was applied to further purify and isolate BSA and IgG. The new procedure produced highly purified BSA and IgG, 98% and 96.8%, respectively, and yielded ideal output, 2.18% and 0.54%, from starting plasma, respectively. The new technique is a rapid and is an available pilot process to prepare the plasma fractions devoid of cellular components.     

A rapid purification method for human erythrocyte pyruvate kinase

Human erythrocyte pyruvate kinase (ATP: pyruvate phosphotransferase, E.C.2.7.1.40) is purified 30,000-fold, using a method which includes ammonium sulfate precipitation, Sephadex G-75 filtration, and Blue Dextran-Sepharose 4B chromatography. The enzyme is resolved into two peaks on Blue Dextran-Sepharose 4B. The first peak with sp act of 300 corresponds to the mature form (R4) whereas the second peak with sp act of 180 corresponds to R2R'2. Peaks I and II give one band on 10% polyacrylamide gel without SDS. Peak II gives two bands on 10% SDS gel with molecular weights 60,000 (R') and 57,500 (R). On the other hand, peak I gives only one band on 10% SDS gel having a molecular weight of 57,500. Both the R4 and R2R'2 forms of the enzyme have the same pH optimum of 7.2.     

The purification and characterization of recombinant human renin expressed in the human kidney cell line 293

The cDNA encoding human preprorenin has been introduced into the adenovirus-transformed human kidney cell line 293. The recombinant 293 cells expressed and secreted prorenin; trypsin was used to activate the secreted prorenin to renin in vitro. The recombinant protein was purified to homogeneity by a single affinity chromatographic step. Using synthetic tetradecapeptide, the Km was 57.1 +/- 9.3 microM and the kcat was (7.48 +/- 1.57) x 10(3)/hr. Activation with trypsin resulted in a secondary cleavage between Arg53 and Leu54 generating a two chain form held together via a disulfide between Cys51 and Cys58. This secondary cleavage did not affect enzyme activity as determined by the ability of renin to degrade a synthetic tetradecapeptide substrate. Our paper demonstrates the potential for producing large quantities of renin from human kidney cells and also suggests that the use of trypsin, which has been widely used to convert prorenin to renin in vitro, causes a secondary cleavage in the renin peptide chain.     

Production and purification of novel secreted human proteins

Our objective during the last year was to produce and purify 50–80 novel, secreted human proteins identified via high throughput cDNA sequencing and computer analysis. We chose the baculovirus expression vector system in order to obtain secreted, correctly folded, bioactive proteins. Recombinant (re-)baculoviruses (BV) were plaque purified, and pulse-labeling was used to verify the synthesis and secretion of the re-proteins. N-terminal microsequencing was performed to simultaneously confirm the identity of the protein(s) as well as the signal peptide (SP) cleavage site(s). Following sequence confirmation, the proteins were purified to homogeneity and functional assays carried out to determine potential therapeutic applications. We identified proteins with antiviral activity, several novel growth factors, proteins influencing the differentiation of specific cell types, novel proteases and protease inhibitors among others. Certain proteins were expressed both in insect cells and in CHO stable cell lines. In the cases analyzed, we found that the same SP cleavage site was utilized in the two expression systems. Significant differences were observed in the carbohydrate moieties attached to the proteins, though no effects on the biological activity due to these differences have been demonstrated. The BV system has served as a viable alternative for the high throughput, high fidelity expression of many novel secreted human genes. To date, more than 75 new genes have been expressed, and the re-proteins purified. This expression system combines many favorable traits including relative speed, moderate cost but perhaps most importantly, the production of biologically active proteins.