Effect of matrices on affinity purification of protein C

One of the critical problems in scale-up of affinity chromatography is the mechanical strength of the support matrix against pressure. Because the costs of both the gel matrix and the ligand for the affinity chromatography are very high, the reusability of gel matrices is directly related to the total production cost. In certain cases, where the source material is viscous (e.g., blood plasma), irreversible deformation of gel matrices can readily occur, necessitating severe constraints in the flow rate. Consequently, productivity is low.We have characterized the system parameters and investigated the performance of various matrices that are commercially available. The experimental system used for this study was the immunoaffinity purification of protein C (an anticoagulant protein) from human blood plasma. The support matrices studied were cross-linked agarose, polymethyl acrylic, cellulose, and polyvinyl alcohol polymers. The major system parameters studied were pressure tolerance, coupling efficiency, adsorption efficiency, and batch adsorption/desorption kinetics of protein C to/from the monoclonal antibody (MAb)-Matrix complex. In addition, the apparent equilibrium constant and bandwidth of the product concentration profile in the eluate were characterized by performing pulse tests.A methodology was developed for evaluating the immunoaffinity colum performance for the separation of protein C. By utilizing the experimentally measured parameters, the flow rate limitation for each purification step was computed. Then, the purification performance of the matrices were evaluated in terms of productivity per unit time. Among the matrices tested, cellulose was superior in overall performance for the immunoaffinity purification of protein C using a 10 cm x 10 cm column.     

Kinetic properties of a L-cysteine desulfhydrase-deficient mutant in the enzymatic formation of L-cysteine from D,L-ATC

Summary A mutant strain lacking in activity of L-cysteine desulfhydrase, a L-cysteine-decomposing enzyme, was screened after UV-treatment ofPseudomonas sp. CU6. The properties of the two strains, original and mutant, were compared on the basis of parameter values estimated from kinetic simulations of the enzymatic formation of L-cysteine from D,L-ATC. Both strains suffered from product inhibition, though inhibition was less for the mutant strain.     

Purification and some properties of a phospholipase A2 from bovine platelets

An intracellular form of phospholipase A2 was purified about 47,500-fold to near homogeneity from bovine platelets 100,000 x g supernatant by sequential use of column chromatographies on Heparin-Sepharose, DEAE-Sephacel, Butyl-Toyopearl, Sephacryl S-300, DEAE-5PW HPLC, TSK G 3000 SW HPLC and Mono Q FPLC. The final preparation showed a single band on SDS-polyacrylamide gel, and its molecular mass was estimated to be approximately 100,000 daltons. The purified PLA2 showed maximal activity at alkaline pH(pH 9.0-10.0) and considerable activity at 0.3-1.0 microM calcium concentration. It hydrolyzed phosphatidylcholine containing arachidonate at sn-2 position with high selectivity in comparison to linoleate.     

Unfolding-refolding of the domains in yeast phosphoglycerate kinase: comparison with the isolated engineered domains

The role of domains as folding units was investigated with a two-domain protein, yeast phosphoglycerate kinase. Each of the domains was produced independently by site-directed mutagenesis. It has been previously demonstrated by several criteria that these domains are able to fold in vivo into a quasi-native structure [Minard et al. (1989a) Protein Eng. 3, 55-60; Fairbrother et al. (1989) Protein Eng. 3, 5-11]. In the present study, the reversibility of the unfolding-refolding process induced by guanidine hydrochloride was investigated for the intact protein and the isolated domains. The transitions were followed by circular dichroism for both domains and the intact protein and by the variations in enzyme activity for the intact protein. Tryptophan residues were used as intrinsic conformational probes of the C-domain. An extrinsic fluorescent probe, N-[[(iodoacetyl)amino]ethyl]-8-naphthylamine-1-sulfonic acid (IAEDANS), was bound to the unique cysteinyl residue Cys97 to observe the conformational events in the N-domain. The unfolding-refolding transitions of each domain in the intact protein and in the isolated domains prepared by site-directed mutagenesis were compared. It was shown that the two domains are able to refold in a fully reversible process. A hyperfluorescent intermediate was detected during the folding of both the isolated C-domain and the intact yeast phosphoglycerate kinase. The stability of each isolated domain was found to be similar, the free energy of unfolding being approximately half that of the intact molecule.     

Effect of operating parameters on specific production rate of a cloned-gene product and performance of recombinant fermentation process

A two-stage continuous system in combination with a temperature-sensitive expression system were used as model systems to maximize the productivity of a cloned gene and minimize the problem associated with the plasmid instability for a high-expression recombinant. In order to optimize the two-stage fermentation process, the effects of such operational variables as temperature and dilution rate on productivity of cloned gene were studied using the model systems and a recombinant, Escherichia coli K12 DeltaH1 Deltatrp/pPLc23trp A1. When the expression of cloned gene is induced by raising the operating temperature above 38 degrees C, a significant decrease in the colony-forming-units (CFU) of the plasmid-harboring cell was observed, and the decrease was related to the product concentration. In order to describe this phenomenon, a new kinetic parameter related to the metabolic stress (metabolic stress factor) was introduced. It is defined as the ratio of the rate of change of pheno-type from colony-forming to non-colony-forming cells to the product accumulation per unit cell mass. At a fixed temperature of 40 degrees C, the varying dilution rate D in the range of 0.35-0.90 h(-1) did not affect the metabolic stress factor significantly. At a fixed dilution rate of D = 0.35 h(-1), this factor remained practically constant up to 41 degrees C but increased rapidly beyond 41 degrees C. The effects of temperature and dilution rate in the second stage on the specific production rate were also studied while maintaining the apparent specific growth rate (mu(2) (app)) of the second stage constant at or near mu(2) (app) = 0.26 h(-1). Under a constant dilution rate, D(2) = 0.35 h(-1), the maximum specific production rate obtained was about q(p, max) = 38 units TrpA/mg cell/h at 41 degrees C. At a constant temperature, T(2) = 40 degrees C, specific production rate increased with decreasing dilution rate with in the dilution rate range of D(2) = 0.35-0.90 h(-1). Based on the results of our study, the optimal operating conditions found were dilution rate D(2) = 0.35 h(-1) and operating temperature T(2) = 41 degrees C at the apparent specific growth rate of 0.26 h(-1). Under the optimal operating conditions, about threefold increase in productivity was achieved compared to the best batch culture result. In addition, the fermentation period could be extended for more than 100 h.     

Studies on quantitative physiology of Trichoderma reesei with two-stage continuous culture for cellulose production

By employing a two-stage continuous-culture system, some of the more important physiological parameters involved in cellulose biosynthesis have been evaluated with an ultimate objective of designing an optimally controlled cellulose process. The two-stage continuous-culture system was run for a period of 1350 hr with Trichoderma reesei strain MCG-77. The temperature and pH were controlled at 32°C and pH 4.5 for the first stage (growth) and 28°C and pH 3.5 for the second stage (enzyme production). Lactose was the only carbon source for the both stages. The ratio of specific uptake rate of carbon to that of nitrogen, Q(C)/Q(N), that supported good cell growth ranged from 11 to 15, and the ratio for maximum specific enzyme productivity ranged from 5 to 13. The maintenance coefficients determined for oxygen, M_O, and for carbon source, M_C, are 0.85 mmol O₂/g biomass/hr and 0.14 mmol hexose/g biomass/hr, respectively. The yield constants determined are: Y_X/O = 32.3 g biomass/mol O₂, Y_X/C = 1.1 g biomass/g C or Y_X/C = 0.44 g biomass/g hexose, Y_X/N = 12.5 g biomass/g nitrogen for the cell growth stage, and Y_X/N = 16.6 g biomass/g nitrogen for the enzyme production stage. Enzyme was produced only in the second stage. Volumetric and specific enzyme productivities obtained were 90 IU/liter/hr and 8 IU/g biomass/hr, respectively. The maximum specific enzyme productivity observed was 14.8 IU/g biomass/hr. The optimal dilution rate in the second stage that corresponded to the maximum enzyme productivity was 0.026 ～ 0.028 hr^?1, and the specific growth rate in the second stage that supported maximum specific enzyme productivity was equal to or slightly less than zero.     

The synthesis,characterization, and preliminary biological evaluation of 1-β-D-arabinofuranosylcytosine-5′-diphosphate-L-1,2-dipalmitin

Recently, 1-β-$\text{D}$-arabinofuranosylcytosine-5′-diphosphate-$\text{DL}$-1,2-dipalmitin (VIa) was reported to inhibit the growth of L51784 cells in mice and of human colon carcinoma HCT-15 cells, also in mice. This paper describes the synthesis of a single diastereomer by conversion of 1-β-$\text{D}$-arabinofuranosylcytosine 5′-monophosphate (II) to the nucleoside 5′-phosphomorpholidate (III), followed by reaction with $\text{L}$-α-dipalmitoylphosphatidic acid (IV) to give 1-β-$\text{D}$-arabinofuranosylcytosine-5′-diphosphate-$\text{L}$-1,2-dipalmitin (V) in good yield. The separation of the product is described and its characterization by chromatography, elemental analysis, and spectroscopic methods. The lipophilic nature of V renders it insoluble in aqueous media and a method of sample preparation utilizing sonication techniques is described which provides a clear solution suitable for biological evaluation. In addition, the ability of V to inhibit the $\text{in}\text{vitro}$ growth of L1210 cells and of mouse myeloma MPC 11 cells is desscribed and compared with 1-β-$\text{D}$-arabinofuranosylcytosine (I) and other lipophilic prodrugs of I.     

Co-amplification of EBNA-1 and PyLT through dhfr-mediated gene amplification for improving foreign protein production in transient gene expression in CHO cells

Applied Microbiology and Biotechnology - Despite the relatively low transfection efficiency and low specific foreign protein productivity (qp) of Chinese hamster ovary (CHO) cell-based transient...