Abstract
A new protein immobilization and purification system has been developed based on the improved plasmid vectors, designated pETChBD-X, which contained the gene coding for two novel chitin-binding domains ChBD-AB, factor Xa cleavage site and adapted for gene fusions. The ChBD-AD from Chitinolyticbacter meiyuanensis SYBC-H1 was used as a novel affinity tag to anchor fusion proteins to chitin granules. The granules carrying the ChBD-AD fusion proteins can be isolated by a simple centrifugation step and used directly for some applications. Moreover, when required, a practically pure preparation of the soluble recombination protein can be obtained after Factor Xa cleavage. The efficiency of this system has been demonstrated by reaching 95% of protein absorbed to chitin within 30 min and recycling over 75% of interest protein after Factor Xa cleavage to separate interest protein and fusion tag. Furthermore, 65% L-glutamate oxidase with this fusion tag could be purified and immobilized within only one step and to be reused in converting L-glutamate to α-ketoglutaric acid directly, the average conversion rate kept above 65% even within four batches of enzyme conversion reaction.
Highlights
Chromatography is an important biological technique used for separating, identifying, and purifying target proteins from a mixture based on different characteristics such as size and shape, total charge, hydrophobic groups present on the surface and so on (Coskun, 2016)
Chitin loaded with immobilized green fluorescent protein (GFP) was suspended again by phosphate buffer solution (PBS) and right amount of Factor-Xa (0.2∼0.3 U/100 μg interest protein) was Analysis of Putative ChBDs From CmChi1
Amino acid sequence analysis revealed that CmChi1 encoded two chitin-binding modules named module A and B, respectively
Summary
Chromatography is an important biological technique used for separating, identifying, and purifying target proteins from a mixture based on different characteristics such as size and shape, total charge, hydrophobic groups present on the surface and so on (Coskun, 2016). A variety of chromatography methods are widely applied in various occasions where protein purification or immobilization are required. By fusing the coding sequence of the interest protein with different tag sequences, which have high affinity to a ligand, these protein can be efficiently purified for identification and characterization (Li et al, 2017). Protein affinity tag is an indispensable tool for recombinant protein expression and purification. Traditional affinity chromatography methods are always high cost and cumbersome operation, it appears important to develop new affinity chromatography methods to overcome these shortcomings
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
