NDLI: Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Content Provider	PubMed Central
Author	Mccormick, Aleesha M. Jarmusik, Natalie A. Endrizzi, Elizabeth J. Leipzig, Nic D.
Copyright Year	2014
Abstract	Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.
Related Links	http://dx.doi.org/10.3791/51295
Starting Page	51295
File Format	PDF
ISSN	1940087X
e-ISSN	1940087X
Journal	Journal of Visualized Experiments : JoVE
Issue Number	83
Language	English
Publisher	MyJove Corporation
Publisher Date	2014-01-01
Access Restriction	Open
Rights Holder	MyJove Corporation
Subject Keyword	Biochemistry, Genetics and Molecular Biology(all) Immunology and Microbiology(all) Chemical Engineering(all) Neuroscience(all) Medicine(all) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Neuroscience Immunology and Microbiology Biochemistry, Genetics and Molecular Biology Chemical Engineering

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4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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