Guhan Jayaraman

Characterization of non-linear adsorption properties of dextran-based polyelectrolyte displacers in ion-exchange systems

Abstract Experimental studies were carried out on the non-linear adsorption properties of dextran-based polyelectrolytes in anion- and cation-exchange chromatographic systems. By monitoring both the induced salt gradients and sequential breakthrough fronts, parameters were determined for use in a Steric Mass Action (SMA) model of non-linear ion-exchange chromatography. These parameters include: total ion capacity of the columns, characteristic charge, steric factor, equilibrium constant, and maximum adsorptive capacity for each of the polyelectrolytes. In addition the number of functional groups were determined by elemental analysis. The values of the SMA parameters were found to be independent of salt and polyelectrolyte bulk phase compositions. Parameters were also determined for a variety of proteins. Experimental isotherms for the polyelectrolytes and proteins were compared with those simulated by the SMA model. Finally, the implications of polyelectrolyte adsorption properties with respect to their ability to act as efficient displacers in ion-exchange displacement systems are discussed.

Ion-exchange displacement chromatography of proteins: Dextran-based polyelectrolytes as high affinity displacerss

Abstract Dextran-based polyelectrolyte displacers were successfully employed for the displacement purification of proteins in ion-exchange displacement systems. The effect of molecular mass was investigated by examining the efficacy of DEAE-dextran and dextran sulfate displacers of various molecular masses in cation- and anion-exchange systems, respectively. Induced salt gradients produced during these displacement experiments were measured in order to study their effect on the protein separations. The unique characteristics of these displacements were well predicted by simulations obtained from a steric mass action (SMA) ion-exchange model. These displacements differ from the traditional vision of displacement chromatography in several important ways: the isotherm of the displacer does not necessarily lie above the feed component isotherms; the concentration of the displaced proteins can sometimes exceed that of the displacer; higher-molecular-mass displacers are not necesarily more efficacious than lower-molecular-mass compounds; and the salt gradients induced by the adsorption of the displacer produce different salt micro-environments for each displaced protein.

Ion-exchange displacement chromatography of proteins Dendritic polymers as novel displacers

While the ability to carry out simultaneous concentration and purification in a single displacement step has significant advantages for downstream processing of biopharmaceuticals, a major obstacle to the implementation of displacement chromatography has been the lack of appropriate displacer compounds. All protein displacement separations reported to date have employed relatively high-molecular-mass (> 2000) polyelectrolyte displacers. In this paper, results are presented on the discovery that low-molecular-mass dendritic polymers can be successfully employed as efficient displacers for protein purification in ion-exchange systems. Pentaerythritol-based dendritic polyelectrolytes ranging in molecular mass from 480 to 5100 were investigated as potential displacers for the purification of proteins in cation-exchange systems. The adsorption properties of these dendrimers were investigated using the steric mass action (SMA) model of non-linear ion-exchange chromatography. An analysis of the resulting SMA parameters using a dynamic affinity plot indicated that these dendrimers should have sufficient affinity to act as protein displacers. Displacement separations of protein mixtures in cation-exchange systems were carried out using zero-, first- and second-generation dendrimers. These experiments demonstrate that this new class of dendritic polyelectrolytes can indeed act as efficient protein displacers. The ability of a low-molecular-mass compound such as the “zero”-generation dendrimer (Mr 480) to displace proteins is very significant in that it represents a major departure from the conventional wisdom that large polyelectrolyte polymers are required to displace proteins in ion-exchange systems. In addition to the fundamental interest generated by low-molecular-mass displacers, it is likely that these displacers will have significant operational advantages as compared to large polyelectrolyte displacers.

Displacement chromatography of proteins using low molecular weight displacers

A major impediment to the implementation of displacement chromatography has been the lack of suitable displacer compounds. Recently, it has been shown that low molecular weight dendritic polymers, protected amino acids and antibiotics can be successfully employed for displacement purification in cation-exchange systems. In this paper, a variety of low molecular weight anionic displacers are identified for the resolution of a bovine β-lactoglobulin mixture into two closely related forms (A and B). A Dynamic Affinity plot is employed to evaluate the affinity of these low molecular weight compounds under various displacement conditions. In contrast to large polyelectrolyte displacers, the efficacy of these low molecular weight displacers are shown to be dependent on displacer concentration. In fact, the Dynamic Affinity Plot qualitatively predicts the transition from a displacement to a desorption regime with these low molecular weight displacers. In addition to the fundamental interest generated by low molecular weight displacers, it is likely that these displacers will have significant operational advantages as compared to large polyelectrolyte displacers. Furthermore, the ability to carry out selective displacement chromatography with these low molecular weight displacers offers significant potential for developing robust large scale displacement processes.

Glycerol conversion to 1, 3-Propanediol is enhanced by the expression of a heterologous alcohol dehydrogenase gene in Lactobacillus reuteri

In this work, Lactobacillus reuteri has been metabolically engineered for improving 1, 3-propanediol (1, 3-PD) production by the expression of an Escherichia coli alcohol dehydrogenase, yqhD, that is known to efficiently convert the precursor 3-hydroxypropionaldehyde (3-HPA) to 1, 3-PD. The engineered strain exhibited significantly altered formation rates for the product and other metabolites during the fermentation. An increase in the 1, 3-PD specific productivity of 34% and molar yield by 13% was achieved in the clone, relative to the native strain. A concomitant decrease in the levels of toxic intermediate, 3-HPA, was observed, with the specific productivity levels being 25% lesser than that of the native strain. Interestingly, the recombinant strain exhibited elevated rates of lactate and ethanol formation as well as reduced rate of acetate production, compared to the native strain. The preferential utilization of NADPH by YqhD with a possible decrease in the native 1, 3-PD oxidoreductase (NADH-dependent) activity, could have resulted in the diversion of surplus NADH towards increased lactate and ethanol productivities.

Transcription analysis of hyaluronan biosynthesis genes in Streptococcus zooepidemicus and metabolically engineered Lactococcus lactis

The has operon genes in the hyaluronan (HA) producer, Streptococcus zooepidemicus, encode for some of the critical enzymes in the HA biosynthetic pathway. Heterologous expression of different combinations of multiple has genes has resulted in increasing HA production to varying degrees in different recombinant strains. In this work, a recombinant Lactococcus lactis strain (SJR6) was constructed, with insertion of three has operon genes (hasABD) from S. zooepidemicus. The SJR6 strain was found to be a better HA producer than two previously constructed recombinant L. lactis strains (SJR2 and SJR3), containing hasAB and hasABC genes, respectively, but exhibited lower HA production than the native HA producer S. zooepidemicus. To understand the differences in HA yield between the various strains, transcriptions of the HA biosynthesis genes (has genes and their homologues) were compared at different phases of exponential growth of the L. lactis and S. zooepidemicus cultures. The mRNA levels of all the heterologous has genes were expectedly far higher than their corresponding homologues in the L. lactis strains. The relative mRNA level of the hasB-homologue, viz. ugd (encoding UDP-glucose dehydrogenase), was found to be much lower than that of other homologues, corroborating earlier reports which indicate tight transcriptional regulation of the ugd gene in L. lactis. Interestingly, all the has gene homologues were found to be up-regulated in all the recombinant L. lactis strains, when compared with the corresponding genes in the untransformed strain, L. lactis NZ9000. A transcription analysis of S. zooepidemicus cultures revealed that the has operon was down-regulated in the mid-exponential growth phase in comparison to the early- and late-exponential growth phases. The transcription analyses in this study have provided insights for the design of recombinant strains with higher HA productivity.

Preparative chromatography in biotechnology.

The field of preparative chromatography has experienced several important advances recently in both the structure of chromatographic materials and modes of chromatographic operation. This review presents recent applications of preparative chromatography in biotechnology, as well as novel stationary phase materials and engineering approaches to preparative chromatographic bioseparations.

Displacement chromatography of biomolecules with large particle diameter systems

Abstract Displacement chromatography was employed for the preparative-scale separation of peptides and proteins using large particle diameter chromatographic systems. Peptide displacements were successfully scaled-up with respect to particle and column diameter with no adverse effects on product recovery. Protein displacements on 30- and 90-μm agarose-based adsorbent systems resulted in well separated displacement zones of pure material. The present work extends the scope of biopolymer displacement chromatography to large particle diameter systems and is expected to further increase the distinct economic advantages associated with preparative-scale displacement chromatography.

Ratio of intracellular precursors concentration and their flux influences hyaluronic acid molecular weight in Streptococcus zooepidemicus and recombinant Lactococcus lactis.

HA molecular weight variation in Streptococcus zooepidemicus and two recombinant Lactococcus lactis strains were investigated by chemostat experiments and metabolic flux analysis (MFA). The study showed that intracellular flux ratio of UDP-GlcUA to UDP-GlcNAc correlated directly with HA molecular weight, for all the three strains. The ratio of intracellular concentration of these HA precursors also exhibited a similar trend. Phosphoglucoisomerase activity and glucose flux towards lactic acid formation were found to be the major bottlenecks for HA production in all the three strains. The study suggests that environmental conditions and genetic manipulations that balance the intracellular flux and HA precursors concentrations will result in increased molecular weight.

Recombinant protein purification using gradient assisted simulated moving bed hydrophobic interaction chromatography. Part II: Process design and experimental validation

In the first part of this work adsorption isotherm parameters were acquired to describe the migration of recombinant streptokinase in Butyl Sepharose columns at different salt concentrations. Based on these results, a simulated moving bed (SMB) chromatographic process was designed and realised, which exploits a two-step salt gradient and allows the continuous separation of streptokinase from contaminants present in a clarified Escherichia coli cell lysate solution. This second part describes the design of the three-zone open-loop gradient SMB process applying both equilibrium theory and an equilibrium stage model and presents results of a series of experiments aiming to obtain pure streptokinase. Moreover, the potential of the SMB process and the design approach are evaluated.