Other methods of protein immobilization

The following protocol for passive adsorption is based on methods reported for use with hydrophobic polymeric particles, such as polystyrene latex beads or copolymers of the same. Other polymer particle types also may be used in this process, provided they have the necessary hydrophobic character to promote adsorption. For particular proteins, conditions may need to be optimized to take into consideration maximal protein stability and activity after adsorption. Some proteins may undergo extensive denaturation after immobilization onto hydrophobic surfaces therefore, covalent methods of coupling onto more hydrophilic particle surfaces may be a better choice for maintaining native protein structure and long-term stability. 

Other methods that are related to affinity chromatography include hydrophobic interaction chromatography and thiophilic adsorption. The former is based on the interactions of proteins, peptides, and nucleic acids with short nonpolar chains on a support. This was first described in 1972 [113,114] following work that examined the role of spacer arms on the nonspecific adsorption of affinity columns [114]. Thiophilic adsorption, also known as covalent or chemisorption chromatography, makes use of immobilized thiol groups for solute retention [115]. Applications of this method include the analysis of sulfhydryl-containing peptides or proteins and mercurated polynucleotides [116]. 

RME shows particular promise in the recovery of proteins/enzymes [12-14]. In the past two decades, the potential of RME in the separation of biological macromolecules has been demonstrated [15-20]. RMs have also been used as media for hosting enzymatic reactions [21-23]. Martinek et al. [24] were the first to demonstrate the catalytic activity of a-chymotrypsin in RMs of bis (2-ethyl-hexyl) sodium sulfosuccinate (Aerosol-OT or AOT) in octane. Since then, many enzymes have been solubilized and studied for their activity in RMs. Other important applications of RME include tertiary oil recovery [25], extraction of metals from raw ores [26], and in drug delivery [27]. Application of RMs/mi-croemulsions/surfactant emulsions were recognized as a simple and highly effective method for enzyme immobilization for carrying out several enzymatic transformations [28-31]. Recently, Scheper and coworkers have provided a detailed account on the emulsion immobiUzed enzymes in an exhaustive review [32]. 

Since the protein scaffold is commonly not very stable, many methods have been used for stabilization presence of additives, immobilization by multiple-point attachment, stabilization by chemical or biochemical modification and by protein engineering, and several others. 

Tuerker and Mavituna immobilized Trichoderma reesei within the open porous networks of reticulated polyurethane foam matrices. Growth pattern, glucose consumption, and cellulase production were compared with those of freely suspended cells. The method of immobilization was simple and had no detrimental effect on cell activity. Hundreds of similar projects could be cited. Not all rated the use of polymethane as the preferred technique. If a statistical analysis were conducted on aU the immobilization literature, we are sure that no single technique would be dominant. However, the combination of ease of immobilization, cost of materials, flow-through properties, control of flux rate through the immobilizing membrane, high surface-to-volume ratio, and other factors make polymethane a viable substratum for the continuous production of proteins. 

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