Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography.
CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016.
Biochemistry and Molecular Biology Program, Michigan Technological University, Houghton, MI, 49931.
Nasrin
N
Salehi
Dept. of Biological Engineering, University of Idaho, Moscow, ID, 83844.
Ching-An
CA
Peng
D000818:Animals; D001710:Biotin / Q000737:chemistry* / Q000378:metabolism; D051928:CD47 Antigen / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry* / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D051928:CD47 Antigen; C497894:CD47 protein, human; D011993:Recombinant Fusion Proteins; D001710:Biotin; D012685:Sepharose; D019809:Streptavidin
CD47; affinity chromatography; biotin; core streptavidin; phagocytosis