The purification of biomolecules is a very challenging process due to the presence of a very complex mixture of other cell culture components. In order to achieve high protein yield and purity, a so-called affinity chromatography step is widely used [1]. In this study, affinity chromatography has been performed through the use of split-inteins. The stationary phase resin is functionalized with a N-intein affinity tag, while the Protein of Interest (POI) has a C-intein tag. The capture of the POI from the crude lysate occurs due to the high affinity between these two tags [2]. The subsequent cleavage causes the release of the purified protein. After the elution, a regeneration step is required in order to get rid of the C-intein tag (see Figure). This stationary affinity phase was tested and characterized. The pore size distribution of the resin was measured by inverse size-exclusion chromatography (iSEC). The binding and saturation capacities were determined through breakthrough curve (BTC) fitting. In the initial purification processes, proteins pre-purified by IMAC (Immobilized Metal Affinity Chromatography) as well as cell lysate were utilized. Then the results were compared and the purification protocol has been optimized for the crude lysate. Afterwards, using a model based optimization [3], both a batch capture process and a two-column CaptureSMB process were simulated in order to predict the best conditions in terms of productivity and column capacity utilization.
Split-Intein Mediated Affinity Chromatography for the Purification of a C-intein Tagged Protein
Simona Felletti
;Alberto Cavazzini;
2018
Abstract
The purification of biomolecules is a very challenging process due to the presence of a very complex mixture of other cell culture components. In order to achieve high protein yield and purity, a so-called affinity chromatography step is widely used [1]. In this study, affinity chromatography has been performed through the use of split-inteins. The stationary phase resin is functionalized with a N-intein affinity tag, while the Protein of Interest (POI) has a C-intein tag. The capture of the POI from the crude lysate occurs due to the high affinity between these two tags [2]. The subsequent cleavage causes the release of the purified protein. After the elution, a regeneration step is required in order to get rid of the C-intein tag (see Figure). This stationary affinity phase was tested and characterized. The pore size distribution of the resin was measured by inverse size-exclusion chromatography (iSEC). The binding and saturation capacities were determined through breakthrough curve (BTC) fitting. In the initial purification processes, proteins pre-purified by IMAC (Immobilized Metal Affinity Chromatography) as well as cell lysate were utilized. Then the results were compared and the purification protocol has been optimized for the crude lysate. Afterwards, using a model based optimization [3], both a batch capture process and a two-column CaptureSMB process were simulated in order to predict the best conditions in terms of productivity and column capacity utilization.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
