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

Abstract

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.

Metal-Linked Dimerization in the Iron-Dependent Regulator from Mycobacterium tuberculosis

“…Metal binding by IdeR represents a competition between the protein and small molecule buffer components for the coordination of the solvated metal ions. HEPES was introduced by Good as a nonchelating buffer (43) and is a preferred buffer for Ni(II)-based immobilized metal affinity chromatography because it has a lower binding affinity for Ni(II) than does Tris, which binds Ni(II) very weakly (44). However, given the low buffer concentration used in this and the previous study (16), buffer coordination would not significantly affect the metal binding equilibrium or alter the sequence of metal binding.…”

A simplified bioprocess for human alpha‐fetoprotein production from inclusion bodies

“…Due to the low process compatibility of guanidine hydrochloride (GuHCl), 6-8 M urea has become the preferred agent for the chemical release of intracellular proteins from E. coli (Choe and Middelberg, 2001;Choe et al, 2002;Falconer et al, 1997Falconer et al, , 1998. However, urea produces cyanate ions, as a result of decomposition in aqueous solution, leading to protein carbamylation and thus product microchemical heterogeneity (Stark et al, 1960;Welles et al, 1971).…”

“…The process potential of chemical extraction has been extended by Choe et al (2002) who demonstrated that proteins extracted at high concentration can be directly coupled to a purification step following a DNA contaminant removal step. Such bioprocess intensification has the potential to improve the economics of inclusion body recovery, and might also speed the economical delivery of new biopharmaceuticals to market.…”

Expanded Beds: A Process Solution for Adsorptive Separations in Waste-Water Treatment

“…The excessive intake of copper may also result in schizophrenia and the Alzheimer kind of diseases [3]- [5]. Copper has a similar adverse effect on marine ecosystem, damaging aquatic life [6]- [8].…”