One step purification procedure of elastase from pancreatic powder by affinity chromatography

Pancreatic elastase was isolated from acetone powder of porcine pancreas by a one step purification procedure on a trialanyl CH-Sepharose 4B affinity column. This column exhibited affinity not only for active elastase but also for trypsin and chymotrypsin which were present in the same pancreatic powder. However, as the extent of affinity toward elastase is considerably higher, the proper conditions were determined with which the adsorbed elastase was isolated in a highly purified form. The yield of elastolytic activity ranged from 60–85% and the purified elastase was shown to be one component by polyacrylamide disc electrophoresis.     

One step purification of chymosin by mixed mode chromatography

Mixed mode Sepharose and Perloza bead cellulose matrices were prepared using various chemistries. These matrices contained hydrophobic (aliphatic and/or aromatic) and ionic (carboxylate or alkylamine) groups. Hydrophobic amine ligands were attached to epichlorohydrin activated Sepharose (mixed mode amine matrices). Hexylamine, aminophenylpropanediol and phenylethylamine were the preferred ligands, on the basis of cost and performance. Other mixed mode matrices were produced by incomplete attachment (0-80%) of the same amine ligands to carboxylate matrices. The best results were obtained using unmodified or partially ligand-modified aminocaproic acid Sepharose and Perloza. High ligand densities were used, resulting in high capacity. Furthermore, chymosin was adsorbed at high and low ionic strengths, which reduced sample preparation requirements. Chymosin, essentially homogeneous by electrophoresis, was recovered by a small pH change. The methods described were simple, efficient, inexpensive and provided very good resolution of chymosin from a crude recombinant source. The carboxylate matrices had the best combination of capacity and regeneration properties. The performance of Sepharose and Perloza carboxylate matrices was similar, but higher capacities were found for the latter. Because it is cheaper and can be used at higher flow rates, Perloza should be better suited to large scale application. High capacity chymosin adsorption was found with carboxymethyl ion exchange matrices, but low ionic strength was essential for adsorption and the purity was inferior to that of the mixed mode matrices. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 45-55, 1997.     

Aspartate transcarbamoylase from Phaseolus aureus. Partial purification and properties

1. Aspartate transcarbamoylase from 4-day-old radicles of Phaseolus aureus was purified 190-fold by (NH₄)₂SO₄ fractionation, DEAE-cellulose and DEAE-Sephadex chromatography and Sephadex-gel filtration. The partially purified enzyme, which required P_i for maximum stability, had an apparent molecular weight of 83000±5000. 2. Uridine nucleotides were found to inhibit the activity; UMP was the most potent inhibitor, followed by UDP and UTP. No other nucleotide was found to affect the enzyme, nor could UMP inhibition be overcome by adding another nucleotide. Aspartate gives a hyperbolic substrate-saturation curve, both with and without UMP. The nucleotide inhibitor is non-competitive with respect to this substrate. Carbamoyl phosphate also yields a hyperbolic substrate-saturation curve in the absence of feedback inhibitor, but when UMP is added a sigmoidal pattern results, and the inhibition is competitive with carbamoyl phosphate. 3. The degree of inhibition by UMP is not affected by p-chloromercuribenzoate, urea, mild heat pretreatment or change in pH over the range 8.5–10.5, but is affected by temperature. 4. The aspartate analogue, succinate, both activates and inhibits the reaction, depending on the concentrations of aspartate and succinate used. 5. Kinetic studies with the partially purified enzyme showed that the K_m for carbamoyl phosphate (0.091 mm) is much lower than that for aspartate (1.7mm). A sequential reaction mechanism was inferred from product-inhibition kinetics, with carbamoyl phosphate binding to the enzyme before aspartate, and the product, carbamoylaspartate, being released ahead of P_i. Initial-velocity studies gave a set of parallel reciprocal plots, compatible with an essentially irreversible step occurring before the binding of aspartate.     

One step purification of glucose-6-phosphate dehydrogenase from brain areas by immunoaffinity chromatography

Glucose-6-phosphate dehydrogenase was purified from rabbit brain cortex using a single immunoaffinity chromatographic step and was contaminated only by a 50 kDa protein. The proteins, separated by SDS-PAGE, were sequenced: the glucose-6-phosphate dehydrogenase was blocked at the N-terminal, the co-eluted protein was similar to -tubulin. Our technique can be applied to purification and sequencing of the enzyme from brain areas or to measure its turnover rate in cultured cells.     

Aerobic purification of hydrogenase from Rhizobium japonicum by affinity chromatography.

We purified active hydrogenase from free-living Rhizobium japonicum by affinity chromatography. The uptake hydrogenase of R. japonicum has been treated previously as an oxygen-sensitive protein. In this purification, however, reducing agents were not added nor was there any attempt to exclude oxygen. In fact, the addition of sodium dithionite to aerobically purified protein resulted in the rapid loss of activity. Purified hydrogenase was more stable when stored under O2 than when stored under Ar. Sodium-chloride-washed hydrogen-oxidizing membranes were solubilized in Triton X-100 and deoxycholate and loaded onto a reactive red 120-agarose column. Purified hydrogenase elutes at 0.36 M NaCl, contains a nickel, and has a pH optimum of 6.0. There was 452-fold purification resulting in a specific activity of 76.9 mumol of H2 oxidized per min per mg of protein and a yield of 17%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed subunits with estimated molecular weights of 65,000 and 33,000. Hydrogenase prepared in this manner was used to raise and affinity purify antibodies against both subunits.     

Single step recombinant human growth hormone (rhGH) purification from milk by peptide affinity chromatography

Recombinant human growth hormone (rhGH) is used for the treatment of several pathologies, most of them related to growth. Although different expression systems can be used for its production, the milk from transgenic cows is one of the most interesting due to the high rhGH level achieved (5 g/L). We have designed and synthesized short peptides (9 or 10 amino acid long) using Fmoc chemistry and studied their ability to purify rhGH from milk once immobilized on an agarose support. Using spiked milk with the hormone as a sample, rhGH was purified with 88% yield and 92% purity in a single step with a fold purification of 4.5. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:999–1005, 2018     

Isolation of phosphoglycerate kinases by affinity chromatography

A variety of Sepharose derivatives containing DL-O-phosphorylserine or adenosine nucleotides with different points of attachment, has been synthesized and tested for affinity to phosphoglycerate kinase. The most effective gels contained periodate-oxidized ATP or ADP bound via the ribose by hydrazone formation to adipoyl-dihydrazo-Sepharose. The effect of pH, magnesium and buffer ions on the binding capacity of the ATP derivative of Sepharose has been examined. Optimal elution of phosphoglycerate kinase was investigated using different combinations of adenosine nucleotides, 3-phosphogylcerate and magnesium ions. A method is presented giving conditions for the purification of phosphoglycerate kinase from different sources (spinach, human erythrocytes, human, rabbit and trout muscle). It includes extract preparation, affinity chromatography and gel filtration. The method is greatly superior to known isolation procedures by virtue of its technical simplicity, excellent yield (85-100%) and reproducability. The capacity of the ATP-ribosyl-adipoyl-dihydrazo-Sepharose was 5 mg phosphoglycerate kinase per 1 g of matrix. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate indicated that the final products are homogeneous. The phosphoglycerate kinases from different sources appear to have the same affinity for this ATP derivative of Sepharose, the same molecular weight and the same specific activity.     

Biosynthesis of galactan by a particulate enzyme preparation from Phaseolus aureus seedlings

A particulate cell-free enzyme system was prepared from Phaseolus aureus shoots. This preparation was able to incorporate [(14)C]galactose from UDP-[(14)C]galactose into a water-soluble polysaccharide, which has a probable molecular weight of at least 4600. The only labelled component detectable in the polymer was shown to be [(14)C]galactose; two labelled oligosaccharides containing only [(14)C]galactose were isolated by partial hydrolysis. The galactan-synthesizing activity of this particulate preparation is maximal at 30 degrees and pH7.1 in the presence of 5.0mm-magnesium chloride and 0.2m-sucrose. Although 3-day-old seedlings were used as a source of enzyme, it appears that 4- or 5-day-old beans contain greater synthetase activity. The enzyme system has an apparent Michaelis constant of 5.8x10(-6)m, and will catalyse the polymerization of galactose residues at the rate of 7.5mmumoles/mg. of protein/min. at a substrate concentration of 9.6mm.     

Trans-N-deoxyribosylase: purification by affinity chromatography and characterization.

trans-N-Deoxyribosylase (EC 2.4.2.6) is usually considered as a single protein catalyzing indifferently the transfer of the deoxyribosyl moiety to and from a purine or a pyrimidine base. Affinity chromatography of an extract from Lactobacillus helveticus with two types of ligands allowed the separation and purification of two distinct trans-N-deoxyribosylases. One catalyzes specifically the deoxyribosyl transfer to and from purine bases exclusively: trans-N-deoxyribosylase-I, the other catalyzes the transfer to and from pyrimidine and purine bases: trans-N-deoxyribosylase-II. A Tris inhibition study showed a markedly different susceptibility of the two enzymes. Preliminary results indicate that the purine-specific enzyme is a polymeric enzyme of molecular weight 86 000 (+/- 4000).     

Gonadotropin receptors. Solubilization and purification by affinity chromatography.

Specific gonadotropin receptors of the rat testis were purified 15,000-fold after detergent extraction by a single affinity chromatography step on agarose coupled to human chorionic gonadotropin. Receptors were eluted in the free form at pH 3.2 and did not aggregate in the absence of detergent. The purified receptors displayed about 50% of the maximum theoretical binding activity, and retained the high binding affinity and hormonal specificity of the original gonadotropin receptors of the cell membrane.     

Single step purification of plasmid DNA using peptide ligand affinity chromatography

Single step affinity chromatography was employed for the purification of plasmid DNA (pDNA), thus eliminating several steps compared with current commercial purification methods for pDNA. Significant reduction in pDNA production time and cost was obtained. This chromatographic operation employed a peptide-monolith construct to isolate pDNA from Escherichia coli (E. coli) impurities present in a clarified lysate feedstock. Mild conditions were applied to avoid any degradation of pDNA. The effect of some important parameters on pDNA yield was also evaluated with the aim of optimising the affinity purification of pDNA. The results demonstrate that 81% of pDNA was recovered and contaminating gDNA, RNA and protein were removed below detectable levels.     

Construction of a modified penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus and purification by immobilized metal affinity chromatography.

The mecA-27r gene, which encodes PBP2a-27r, was modified by site-specific mutagenesis, resulting in replacement of the N-terminal membrane anchor with a short chelating peptide (CP-PBP2a-27r). CP-PBP2a-27r retained the same binding affinity for beta-lactam antibiotics as the wild-type enzyme. Approximately 95% pure CP-PBP2a-27r was recovered in a single step by use of chelating-peptide-immobilized metal ion affinity chromatography.     

Rapid purification of mitochondrial hexokinase from rat brain by a single affinity chromatography step on Affi-Gel blue

The mitochondrial hexokinase from rat brain, selectively released from mitochondria by the action of glucose 6-phosphate, can be purified to greater than 90% homogeneity by a single affinity chromatography step on Affi-Gel Blue; the Cibacron Blue F3GA ligand bound to this matrix serves as an analog of ATP, the normal substrate for the enzyme, and selective elution is accomplished using glucose 6-phosphate which is a competitive ligand vs. ATP. With this and other modifications to the previously described procedure highly purified enzyme is readily obtained in good yield and with retention of the ability to rebind to mitochondria.     

Pentose metabolism in Mycobacterium smegmatis: comparison of L-arabinose isomerases induced by L-arabinose and D-galactose.

D-Galactose, which did not serve as a growth substrate, was found to induce an L-arabinose isomerase of similar properties to the L-arabinose-induced L-arabinose isomerase. In both cases the pH profiles, pH stability, optimum temperature, heat stability, substrate specificity, metal ion requirements, mobility on polyacrylamide gel electrophoresis, and kinetic properties of the induced isomerases were identical. It appears possible that D-galactose was incorporated into the cells by an L-arabinose permease system that was alos induced by D-galactose.     

The purification of beta-galactosidase-specific polysomes by affinity chromatography.

Affinity chromatography on a β-galactosidase substrate analog-Sepharose column was used to purify β-galactosidase-specific polysomes from E. coli. The purification was monitored by hybridization of [³H]uridine pulse-labeled RNA extracted from polysomes to p lac 5 DNA. A purification of at least 12-fold was obtained. Binding of lac polysomes to the column required the presence of Sepharose-bound substrate analog; salt and pH conditions favorable to β-galactosidase binding; and intact polyribosomes. It was calculated that 40–50% of the labeled mRNA recovered was lac RNA.     

One step purification of corilagin and ellagic acid from Phyllanthus urinaria using high-speed countercurrent chromatography

High-speed countercurrent chromatography (HSCCC) has been successfully applied to the preparative separation of corilagin and ellagic acid in one step from the Chinese medicinal plant Phyllanthus urinaria L. by use of direct and successive injections of a crude methanolic extract. Some aspects concerning the practical use of this technique in the described application are considered.