Immobilised metal affinity chromatography of β-galactosidase from unclarified Escherichia coli homogenates using expanded bed adsorption

Clemmitt, R. H.; Chase, Howard A.

doi:10.1016/s0021-9673(00)00087-x

Cited by 46 publications

(26 citation statements)

References 52 publications

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“…The efficient adsorption of a product at any particular zone within the expanded bed also depends on the size of the molecule. The significance of the size of the product molecule on binding efficiency has been noted for b-galactosidase (Clemmitt and Chase, 2000), ADH (Gardner et al, 2004) and lysozyme (Bruce and Chase, 2001). Thus the adsorption of a target molecule in any particular zone within an expanded bed will depend on the size of the product, the concentration of the product in the feed and the amount of intact and partially broken cells in the feed that could potentially block the binding sites.…”

Section: Introductionmentioning

confidence: 99%

“…Ion exchange adsorbents are used early in the downstream process because of their high capacity for proteins and enzymes, their general applicability and their resilience to clean-in-place (CIP) procedures. Since electrostatic interactions are involved in the adsorption process, the ionic strength of the feedstock plays a significant role in the adsorption process (Chang and Chase, 1996;Clemmitt and Chase, 2000) which, in turn, is affected by the extent of cell disruption and by the amount and nature of the contaminant molecules present.…”

Section: Introductionmentioning

confidence: 99%

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A study of the influence of yeast cell debris on protein and α‐glucosidase adsorption at various zones within the expanded bed using In‐Bed sampling

Balasundaram

Harrison

et al. 2007

Biotech & Bioengineering

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Expanded bed adsorption chromatography is used to capture products directly from unclarified feedstocks, thus combining solid-liquid separation, product concentration and preliminary purification into a single step. However, when non-specific ion-exchangers are used as the adsorbent in the expanded bed, there is the possibility that electrostatic interactions of cells or cell debris with the adsorbent may interfere with the adsorption of soluble products. These interactions depend on the particle size of the cell debris and its surface charge, which in turn depend on the extent of disruption used to release the intracellular products. The interactions occurring during expanded bed adsorption between the anionic ion-exchanger STREAMLINE DEAE and particulate yeast homogenates obtained by high pressure homogenisation at different intensities of disruption achieved by operating at different pressures were studied, while maintaining all other parameters constant. In-bed sampling from the expanded bed using ports fitted up the height of expanded bed was used to study the retention of yeast cells and cell debris within the bed and its influence on the adsorption of total soluble protein and alpha-glucosidase within various zones of the expanded bed. The retention of the biomass present in the homogenate obtained at a lower intensity of disruption was found to be high at the lower end of the column (17% from 13.8 MPa sample compared to 1% from 41.4 MPa sample). This interaction of the particulate material with the adsorbent was found to reduce the dynamic binding capacity of the adsorbent for total soluble protein from 3.6 mg/mL adsorbent for 41.4 MPa sample to 3.0 mg/mL adsorbent for 13.8 MPa sample. The adsorption of alpha-glucosidase was found to increase with an increase in the concentration of the enzyme in the feed, which increased with the intensity of disruption. Selective adsorption of 6,732 U alpha-glucosidase per mg of total protein bound, was noticed for the feedstock prepared at a higher disruption intensity at 41.4 MPa compared to adsorption of 1,262 U/mg of total protein bound for that prepared at 13.8 MPa. The selective adsorption of alpha-glucosidase due to its high concentration together with simultaneous high specific activity of the enzyme in the feed indicated the significance of selective release of enzymes during microbial cell disruption for efficient expanded bed adsorption processes.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A study of the influence of yeast cell debris on protein and α‐glucosidase adsorption at various zones within the expanded bed using In‐Bed sampling

Balasundaram

Harrison

et al. 2007

Biotech & Bioengineering

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“…They can therefore be separated from contaminating proteins with no or lower affinity for the metals. IMAC can also be performed in denaturing buffers and offers higher selectivity than other chromatographic modes such as anion exchange (Clemmitt et al, 2000b). By performing the L1 purification in expanded mode, very little conditioning is required to remove cells or cell debris between extraction and recovery steps (Chase, 1994;Draeger and Chase, 1991).…”

Section: Introductionmentioning

confidence: 99%

Coupling of chemical extraction and expanded‐bed adsorption for simplified inclusion‐body processing: Optimization using surface plasmon resonance

Choe

Clemmitt

Chase

et al. 2002

Biotech & Bioengineering

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Integration of the chemical extraction of recombinant inclusion-body protein from Escherichia coli, and its recovery by metal-affinity expanded-bed adsorption (IMAC-EBA) under denaturing conditions, was investigated. The viral coat protein L1 with a hexa-histidine tag was expressed in Escherichia coli HMS174(DE3) as a model protein. Interference of released host DNA with adsorbent fluidization in the EBA step was solved by selective precipitation using spermine and low-speed centrifugation. However, the capacity and selectivity of the adsorbent for L1 remained lower than anticipated. The binding of L1 to immobilized Ni(2+) was therefore studied in detail using surface plasmon resonance (SPR). The Tris buffer and ethylene-diamine tetraacetic acid (EDTA) used in the extraction mixture were found to interfere significantly with the L1-Ni(2+) interaction. The SPR studies suggest that L1 binding could be improved by replacing the Tris buffer with HEPES and by adding CaCl(2) to inactivate the EDTA. The modified chemical extraction conditions resulted in effective L1 extraction from cytoplasmic inclusion bodies, at high cell density (OD(600 )= 80) and without the use of reducing agent, into a medium optimized for subsequent IMAC recovery. The modified buffer conditions resulted in an improved binding capacity and a good L1 purification factor (12.7) and recovery yield (71%). This work demonstrates that it is possible to reduce the complexity and hence the cost associated with traditional processes used to prepare purified denatured protein, ready for refolding, from cytoplasmic inclusion bodies.

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“…Ion-exchange EBA chromatography Expanded bed adsorption reduces the need for extensive filtration and/ or centrifugation and concentration steps and has the advantage over other contacting methods of near plug flow of liquid resulting in adsorption performance similar to packed beds [11]. Developments in EBA chromatography technology have enabled Shepard et al [37] to load crude feedstock directly to columns, with no prior treatment, in the routine large-scale cGMP manufacturing of two different human recombinant proteins each secreted by the methylotrophic yeast Pichia pastoris.…”

Section: Chemical Extractionmentioning

confidence: 99%

The economics of inclusion body processing

Lee

Cooney

Middelberg

et al. 2006

Bioprocess Biosyst Eng

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Many recombinant proteins are often over-expressed in host cells, such as Escherichia coli, and are found as insoluble and inactive protein aggregates known as inclusion bodies (IBs). Recently, a novel process for IB extraction and solubilisation, based on chemical extraction, has been reported. While this method has the potential to radically intensify traditional IB processing, the process economics of the new technique have yet to be reported. This study focuses on the evaluation of process economics for several IB processing schemes based on chemical extraction and/or traditional techniques. Simulations and economic analysis were conducted at various processing conditions using granulocyte macrophage-colony stimulating factor, expressed as IBs in E. coli, as a model protein. In most cases, IB processing schemes based on chemical extraction having a shorter downstream cascade demonstrated a competitive economic edge over the conventional route, validating the new process as an economically more viable alternative for IB processing.

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Immobilised metal affinity chromatography of β-galactosidase from unclarified Escherichia coli homogenates using expanded bed adsorption

Cited by 46 publications

References 52 publications

A study of the influence of yeast cell debris on protein and α‐glucosidase adsorption at various zones within the expanded bed using In‐Bed sampling

A study of the influence of yeast cell debris on protein and α‐glucosidase adsorption at various zones within the expanded bed using In‐Bed sampling

Coupling of chemical extraction and expanded‐bed adsorption for simplified inclusion‐body processing: Optimization using surface plasmon resonance

The economics of inclusion body processing

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