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

Summary

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

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.

Introduction

Proteins cover a wide-range of biomolecules that are responsible for many biological functions, ultimately leading to proper tissue formation and organization. These molecules initiate thousands of signaling pathways that control up-regulation and/or down-regulation of genes and other proteins, maintaining equilibrium within the human body. Disruption of a single protein affects this entire web of signals, which can lead to the onset of devastating disorders or diseases. Engineering individual proteins in the lab offers one solution for combating these adverse effects and offers an alternative to small molecule drugs. In 1977, a gene encoding the 14 amino acid somatos....

Protocol

1. Designing of Target Protein

1. Using the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/) obtain an amino sequence for the target protein taking into account the species of interest and any splice variants. Select the amino acid sequence that corresponds to the active region of interest of the protein.
   Note: For Sema3A, amino acids 21-747 were chosen. A fusion protein was designed with NGF where the sequence of barstar, amino acid 1-90, was added to NGF amino acids 122-241.
2. To the N-terminus add the follow sequences: 6X-His tag for purification (HHHHHH), Tobacco Etch Virus (TEV) protease....

Results

Cloning and Test Expression

When plating is performed properly, single isolated colonies should form to increase the chances of plucking clonal transformed bacteria cells (Figure 2A). However, if too many cells are plated, plates are incubated too long at 37 °C or transformation is questionable, colonies may cover the agar plate or form bigger aggregates of cells (Figures 2B and 2C). During test expression, NGF and Sema3A were.......

Discussion

Recombinant protein engineering is a very powerful technique that spans many disciplines. It is cost-effective, tunable and a relatively simple procedure, allowing the production of high yields of custom-designed proteins. It is important to note that designing and expressing target proteins is not always straightforward. Basal expression and recombinant protein stability depend on specific choices of vector, E. coli cell strains, peptide tag additions and cultivation parameters. Our specific design criterion ut.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,4-Dithio-DL-threitol, DTT, 99.5%	Chem-Impex International	127	100 g
2-Hydroxyethylmercaptan β-Mercaptoethanol	Chem-Impex International	642	250 ml
Acetic acid, glacial	EMD	AX0073-9	2.5 L
Agar	Bioshop	AGR001.500	500 g
Ampicillin sodium salt	Sigma-Aldrich	A9518	25 g
Antifoam 204	Sigma-Aldrich	A6426	500 g
Barstar-NGF pET-21a(+)	GenScript USA Inc.		4 µg
BL21(DE3) Competent Cells	Novagen	69450	1 ml; Expression Host
Bradford reagent	Sigma-Aldrich	B6916	500 ml
BugBuster	Novagen	70922-3	100 ml
Gel filtration standard	Bio-Rad	151-1901	6 vials
Glycerol	Bioshop	GLY001.1	1 L
Guanidine hydrodioride amioformamidine hydrochloride	Chem-Impex International	152	1 kg
His-Pur Ni-NTA Resin	Thermo Scientific	88222	100 ml
Hydrochloric acid	EMD	HX0603-3	2.5 L
Imidazole	Chem-Impex International	418	250 g
IPTG	Chem-Impex International	194	100 g
Laemmli sample buffer	Bio-Rad	161-0737	30 ml
Lauryl sulfate sodium salt, Sodium dodecyl surface	Chem-Impex International	270	500 g
LB Broth	Sigma-Aldrich	L3022	1 kg
NovaBlue Competent Cells	Novagen	69825	1 ml; Cloning Host
Phosphate buffered saline	Sigma-Aldrich	P5368-10PAK	10 pack
Potassium Chloride	Chem-Impex International	01247	1 kg
Sema3A-pET-21a(+)	GenScript USA Inc.		4 µg
SimplyBlue SafeStain	Invitrogen	LC6060	1 L
Sodium chloride	Sigma-Aldrich	S5886-1KG	1 kg
Sodium hydroxide	Fisher Scientific	S318-500	500 g
Sodium phosphate diabasic	Sigma-Aldrich	S5136-500G	500 g
Sodium phosphate monobasic	Sigma-Aldrich	S5011	500 g
Terrific Broth	Bioshop	TER409.5	5 kg
Tetracycline hydrochloride	Chem-Impex International	667	25 g
Tris/Glycine/SDS Buffer, 10x	Bio-Rad	1610732	1 L
Trizma Base	Sigma-Aldrich	T1503	1 kg
Tryptone, pancreatic	EMD	1.07213.1000	1 kg
Yeast extract, granulated	EMD	1.03753.0500	500 g
ÄKTApurifier10	GE Healthcare	28-4062-64	Includes kits and accessories
Benchtop Orbital Shaker	Thermo Scientific	SHKE4000	MAXQ 4000
BirA500	Avidity	BirA500	Enzyme comes with reaction buffers and biotin solution
Dialysis Casette	Thermo Scientific	66380	Slide-A-Lyzer (Extra Strength)
Dialysis Tubing	Spectrum Laboratories	132127, 132129	MWCO: 25,000 and 50,000
Flow Diversion Valve FV-923	GE Healthcare	11-0011-70
FluoReporter Biotin Quantification Assay Kit	Invitrogen	1094598
Frac-950 Tube Racks, Rack C	GE Healthcare	18-6083-13
Fraction Collector Frac-950	GE Healthcare	18-6083-00	Includes kits and accessories
Heated/Refrigerated Circulator	VWR	13271-102	Model 1156D
Heating Oven FD Series	Binder		Model FD 115
HiLoad 16/60 Superdex 200 pg	GE Healthcare	17-1069-01	Discontinued--Replacement Product: HiLoad 16/600 Superdex 200 pg
J-26 XPI Avanti Centrifuge	Beckman Coulter	393126
JA 25.50 Rotor	Beckman Coulter	363055
JLA 8.1 Rotor	Beckman Coulter	969329	Includes 1 L polyporpylene bottles
JS 5.3 Rotor	Beckman Coulter	368690
Laminar Flow Hood	Themo Scientific	1849	Forma 1800 Series Clean Bench
Microplate Reader	TECAN		infinite M200
Mini-PROTEAN Tetra Cell	Bio-Rad	165-8004	4-gel vertical electrophoresis system
Mini-PROTEAN TGX Precast Gels	Bio-Rad	456-9036	Any kDa, 15-well comb
Ni-NTA Column	Bio-Rad	737-2512	49 ml volume ECONO-Column
Plasmid Miniprep Kit	Omega Bio-Tek	D6943-01
PowerPac HC Power Supply	Bio-Rad	164-5052	250 V, 3 A, 300 W
Round Bottom Polypropylene Copolymer Tubes	VWR	3119-0050	50 ml tubes for JA 25.50 rotor
Spin-X UF Concentrators	Corning	431488, 431483	20 and 6 ml; MWCO: 10,000 Da
Subcloning Service	GenScript USA Inc.		Protein Services
Ultrasonic Processor	Cole-Parmer	18910445A	Model CV18
Vortex-Genie 2	Scientific Industries	SI-0236	Model G560