An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons

Summary

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells in a cost-effective manner and is purified by affinity chromatography. BDNFavi is then directly mono-biotinylated with the enzyme BirA in a tube. BDNFavi and mono-biotinylated BDNFavi retain their biological activity when compared to commercially available BDNF.

Abstract

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A reproducible protocol was developed to directly mono-biotinylate BDNFAvi with the enzyme BirA in a tube. In this reaction, mono-biotinylated BDNFAvi retains its biological activity.

Neurotrophins are target-derived growth factors playing a role in neuronal development and maintenance. They require rapid transport mechanisms along the endocytic pathway to allow long-distance signaling between different neuronal compartments. The development of molecular tools to study the trafficking of neurotrophins has enabled the precise tracking of these proteins in the cell using in vivo recording. In this protocol, we developed an optimized and cost-effective procedure for the production of mono-biotinylated BDNF. A recombinant BDNF variant containing a biotinylable avi sequence (BDNFAvi) is produced in HEK293 cells in the microgram range and then purified in an easily scalable procedure using affinity chromatography. The purified BDNF can then be homogeneously mono-biotinylated by a direct in vitro reaction with the enzyme BirA in a tube. The biological activity of the mono-biotinylated BDNF (mbtBDNF) can be conjugated to streptavidin-conjugated to different fluorophores. BDNFAvi and mbtBDNF retain their biological activity demonstrated through the detection of downstream phosphorylated targets using western blot and activation of the transcription factor CREB, respectively. Using streptavidin-quantum dots, we were able to visualize mbtBDNF internalization concomitant with activation of CREB, which was detected with a phospho-CREB specific antibody. In addition, mbtBDNF conjugated to streptavidin-quantum dots was suitable for retrograde transport analysis in cortical neurons grown in microfluidic chambers. Thus, in tube produced mbtBDNF is a reliable tool to study physiological signaling endosome dynamics and trafficking in neurons.

Introduction

Neurons are the functional units of the nervous system possessing a complex and specialized morphology that allows synaptic communication, and thus, the generation of coordinated and complex behavior in response to diverse stimuli. Neuronal projections such as dendrites and axons are critical structural features involved in neuronal communication, and neurotrophins are crucial players in determining their morphology and function¹. Neurotrophins are a family of secreted growth factors that include NGF, NT-3, NT-4, and brain-derived neurotrophic factor (BDNF)². In the central nervous system (CNS), BDNF participates in dive....

Protocol

All experiments were carried out in accordance with the approved guidelines of CONICYT (Chilean National Commission for Scientific and Technological Research). The protocols used in this study were approved by the Biosecurity and Bioethical and Animal Welfare Committees of the Pontificia Universidad Católica de Chile. Experiments involving vertebrates were approved by the Bioethical and Animal Welfare Committee of the Pontificia Universidad Católica de Chile.

NOTE: The following protocol was designed to purify BDNFAvi from a total volume of 500 mL of conditioned medium produced in HEK293 cells. The amount of conditioned medium tha....

Results

The use of a chromatographic column-based protocol allows the processing of significant volumes of HEK293 conditioned media. In Figure 1, the results of the purification of BDNFAvi from 500 mL of conditioned media are shown. Consecutive elutions of BDNFAvi from the Ni-NTA agarose beads yield decreasing concentrations of BDNFAvi (Figure 1A). After four consecutive elutions (each lasting 15 min), the majority of the BDNF captured by the beads is recovered. The con.......

Discussion

In this article, an optimized methodology for the production and purification of mbtBDNF in an affinity chromatography-based procedure is described, based on the work of Sung and collaborators¹⁷. The optimizations include the use of a cost-effective transfection reagent (PEI) while maintaining the efficiency of more expensive transfection methods such as lipofectamine. This optimization translates into a significant cost reduction in the protocol, allowing for scalability while maintaining high co.......

Materials

Name	Company	Catalog Number	Comments
2 way stopcock	BioRad	7328102	Chromatography apparatus component
2-mercaptoethanol	Sigma	M6250	BDNF elution buffer
Acrylamide/Bisacrylamide	BioRad	1610154	SDS-PAGE gel preparation
Amicon Ultra-15 10K	Millipore	UFC901024	BDNF concentration
Ammonium Persulfate	Sigma	A9164	SDS-PAGE gel preparation
anti B-III-Tubulin antibody	Sigma	T8578	Western blot assays for BDNF biological activity detection
anti BDNF antibody	Alomone	AGP-021	Western blot assays for BDNF quantification
anti BDNF antibody	Alomone	ANT-010	Western blot assays for BDNF quantification
Anti ERK antibody	Cell Signaling	9102	Western blot assays for BDNF biological activity detection
anti pCREB antibody (S133)	Cell Signaling	9198	Western blot assays for BDNF biological activity detection
anti pERK antibody (T202, Y204)	Cell Signaling	4370	Western blot assays for BDNF biological activity detection
anti pTrkB antibody (Y515)	Abcam	ab109684	Western blot assays for BDNF biological activity detection
Antibiotic/Antimycotic	Gibco	15240-062	HEK293 maintenance
ATP	Sigma	A26209	BDNF monobiotinylation buffer
B-27 Supplement	Gibco	17504-044	Neuron maintenance
Bicine	Sigma	B3876	BDNF monobiotinylation buffer
BirA-GST	BPS Bioscience	70031	Enzyme for BDNF AviTag monobiotinylation
Bovine Fetal Serum	HyClone	HC.SH30396.02	HEK293 maintenance
Bovine Serum Albumin	Jackson ImmunoResearch	001-000-162	BDNF buffer modification component, blocking buffer for western blot and immunofluorescence
D-Biotin	Sigma	B4639	BDNF monobiotinylation buffer
Dithiothreitol	Invitrogen	15508-013
DMEM High Glucose Medium	Gibco	11965-092	Neuron seeding
DMEM Medium	Gibco	11995-081	HEK293 maintenance
Econo Column Funnel	BioRad	7310003	Chromatography apparatus component
EDTA	Merck	108418
EZ-ECL Kit	Biological Industries	1633664	Protein detection by western blotting
Glutamax	Gibco	35050-061	Neuron and HEK293 maintenance
Glycerol	Merck	104094	BDNF elution buffer, lysis buffer for western blot assays
Hettich Rotina 46R Centrifuge	Hettich	Discontinued	Centrifuge used for clearing the medium of debris
Hettich Universal 32R Centrifuge	Hettich	Discontinued	Centrifuge used for protein concentrator centrifugation
Horse Serum	Gibco	16050-122	Neuron seeding
ImageQuant LAS 500	GE Healthcare Life Sciences	29005063	Western blot image acquisition
Imidazole	Sigma	I55513	BDNF buffer modification component
KCl	Winkler	BM-1370	PBS component
KH2PO4	Merck	104873	PBS component
Laminin	Invitrogen	23017-015	Cover coating for compartmentalized neurons
Luer Tubing Adaptor	BioRad	7323245	Chromatography apparatus component
Luminata™ Forte Western HRP Substrate	Millipore	WBLUF0100	Protein detection by western blotting
Mg(CH3COO)2	Merck	105819	BDNF monobiotinylation buffer
Mowiol 4-88	Calbiochem	475904	Mounting reagent for immunofluorescence assays
MyOne C1 Streptavidin Magnetic Beads	Invitrogen	65001	Biotinylation verification
Na2HPO4	Merck	106586	BDNF buffer modification component
NaCl	Winkler	BM-1630	PBS component, BDNF buffer modification component
NaH2PO4	Merck	106346	BDNF buffer modification component
Neurobasal Medium	Gibco	21103-049	Neuron maintenance
Ni-NTA Agarose Beads	Qiagen	30210	BDNF AviTag purification
Nikon Ti2-E	Nikon		Microscope for fluorescence imaging
Nitrocellulose Membrane	BioRad	1620115	Protein transfer for western blotting
ORCA-Flash4.0 V3 Digital CMOS camera	Hamamatsu	C13440-20CU	Camera for epifluorescence imaging
P8340 Protease Inhibitor Cocktail	Sigma	P8340	BDNF buffer modification component
Paraformaldehyde	Merck	104005	Fixative for immunofluorescence assays
Penicillin/Streptomycin	Gibco	15140-122	Neuron maintenance
Poli-D-Lysine	Corning	DLW354210	Cover coating for compartmentalized neurons
Poli-L-Lysine	Millipore	P2363	Cover coating for non-compartmentalized neurons
Poly-Prep Chromatography Column	BioRad	7311550	Chromatography apparatus component
Polyethyleneimine 25K	Polysciences Inc.	PLY-0296	HEK293 transfection
Quantum Dots 655 streptavidin conjugate	Invitrogen	Q10121MP	Monobiotinylated BDNF AviTag label for live and fixed cell experiments
Saponin	Sigma	S4521	Detergent for immunofluorescence assays
Sucrose	Merck	107687
Syldgard 184 silicone elastomer base	Poirot	4019862	Microfluidic chamber preparation
TEMED	Sigma	T9281	SDS-PAGE gel preparation
Tris	Winkler	BM-2000	Lysis buffer component
Triton X100	Merck	108603	Cell permeabilization in immunofluorescence and western blot assays
Trypsin-EDTA 0.5%	Gibco	15400-054	HEK293 passaging