Efficient expression and purification of tag-free recombinant human procalcitonin (hPCT) with precise sequence in E. coli
Protein expression and purification(2024)
摘要
We present an efficient method for expression and purification of recombinant human procalcitonin (hPCT) in E. coli T7 express LysY/Iq cells, ensuring precise N-and C-terminal amino acid sequences. Our method involves fusing codon-optimized cDNA with two distinct tag sequences: eXact tag and chitin binding domain (CBD) tag. To purify the protein, we employ a two-step affinity chromatography process. Firstly, we utilize the N-terminal Profinity eXact tag and purify the protein through Profinity eXact-affinity column chromatography using a resin on which a mutant subtilisin protease was immobilized. The eXact tag was removed by adding NaF to activate the enzyme. Subsequently, the digested sample containing C-terminal CBD tag is directly loaded for the second step of chitin affinity chromatography. Elution is achieved through dithiothreitol (DTT)-catalyzed self-cleavage of the intein sequence from the fusion protein. As a result, the target protein is selectively recovered in the flow -through, completely tag-free, with a purity exceeding 95%. To ensure high purity and eliminate potential con-taminants, we effectively remove E. coli host DNA and endotoxins through a combination of streptomycin sulfate, Triton X-114, and ammonium sulfate treatment. The exceptional level of purity obtained eliminates the need for further purification steps in most applications. This highly purified hPCT can be used as a calibrator in pro-calcitonin or calcitonin immunoassays. Notably, our approach effectively manages small peptides that are prone to degradation by E. coli host proteases, offering a robust solution for various research and application requirements.
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关键词
Human procalcitonin,Escherichia coli,Soluble protein expression,Affinity tag -based protein purification
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