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Creative Enzymes is a biotech company specialized in multiple custom enzyme services. We have decades of experiences in enzyme production and purification for both academic and industrial projects. Our services have received commendations from thousands of customers. With the state-of-art purification strategies for recombinant enzymes, products will be delivered in a short timeframe and high quality according to your needs. In addition, Creative Enzymes is motivated to work closely with customers in case of any special requirements.
Escherichia coli have been most widely used for the production of recombinant enzymes. However, high-level expression in E. coli often results in accumulated enzymes as insoluble aggregates as inclusion bodies. Inclusion body enzymes are devoid of biological activity and need solubilization, refolding, and purification procedures to recover their function. The first step is the purification of inclusion bodies. Afterwards, the insoluble enzyme pellet can be washed with buffer, such as guanidine hydrochloride or urea. Then the washed inclusion bodies are resuspended and incubated in buffer containing a strong denaturant and certain reducing agents. In general, inclusion bodies are solubilized by the use of a high concentration of denaturants, along with the reducing agent. Solubilized enzymes are then refolded by slow removal of the denaturant in the presence of oxidizing agents. Enzyme solubilization from the inclusion body using high concentrations of chaotropic reagents results in the loss of secondary structure leading to the random coil formation of the enzyme structure and exposure of the hydrophobic surface. Loss of secondary structure during solubilization and the interaction among the denatured enzyme molecules during refolding resulting in their aggregation are considered to be the main reasons for the poor recovery of bioactive enzymes from the inclusion bodies.
Figure: Transmission electron micrographs of E. coli cells producing fusion protein of β-gal-insulin chain A (x17,500). Arrow indicates distinct inclusion bodies of fusion protein.
Reference: Mukhopadhyay, Asok. Biotreatment, Downstream Processing and Modelling. Springer Berlin Heidelberg, 1997. 61-109.
Creative Enzymes relies on the expertise in biochemistry and structural biology to simplify the laborious and expensive process of solubilization of inclusion bodies by conventional methods. Our purification services present multiple advantages:
Creative Enzymes promises to deliver biologically active enzymes in a timely and economical fashion. Feel free to contact us for more information about our technology and service.
