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

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.

Covalent growth factor tethering to direct neural stem cell differentiation and self-organization

“…[25] The amino acid sequence was optimized for E. coli expression (Genscript) and inserted into a pET28a vector. Myristoyl-CoA:protein N-myristoyltransferase (CaNMT) inserted into a pET11c vector was generously obtained from Dr. Edward Tate (Imperial College London).…”

“…CaNMT and azIFN- γ were co-expressed within E. coli and purified as previously described. [11, 25] Briefly, BL21 (DE3) E. coli cultures were grown in 1.8 L of 47.6 mg/mL Terrific Broth (EMD) with 8 mL/L glycerol (Sigma-Aldrich), 200 μ g/mL ampicillin (Chem-Impex), and 50 μ g/mL kanamycin (Chem-Impex). After the cultures reached an optical density of 0.7, 500 μ M 12-ADA was added (for no azide tag controls, 12-ADA was omitted) and expression of both proteins induced by 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Chem-Impex) and maintained at 37 °C for 4 h. The culture medium was centrifuged and stored at −80°C until isolation.…”

A Hydrogel Bridge Incorporating Immobilized Growth Factors and Neural Stem/Progenitor Cells to Treat Spinal Cord Injury

“…Recombinant Protein Expression and Purification : Growth factors (IFN‐γ, PDGF‐AA) were expressed, biotinylated, and purified as described in previous studies . Briefly, active sequences for each protein were selected from National Center for Biotechnology Information (NCBI; IFN‐γ: NP_620235 AA23‐156; PDGF‐AA: AAH61731.1, AA87‐211).…”

Neural stem cell encapsulation and differentiation in strain promoted crosslinked polyethylene glycol-based hydrogels

“…The cytokine IFN-c was recombinantly expressed and purified in house as described in previous studies. 43,44 To encourage cell attachment and to increase the local growth factor functional efficiency within the hydrogel, IFN-c protein and laminin were reacted with azide-PEG4-NHS ester at a molar ratio of 1:20 for 3 h to add azide groups to the proteins to create azide-INF-c and azide-laminin ( Figure 1). This nonspecific form of modification targets primary amine containing amino acid residues.…”