The authors would like to acknowledge The University of Akron for the funding that supported this work.

1. Designing of Target Protein
<ol>
	<li>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.</li>
	<li>To the N-terminus add the follow sequences: 6X-His tag for purification (HHHHHH), Tobacco Etch Virus (TEV) protease cut site for removal of His tag (ENLYFQG), biotin tagged sequence for biotinylation of protein at K (GLNDIFEAQKIEWHE) and flexible hinge with gaps that will allow room for proper protein refolding (EFPKPSTPPGSSGGAP). 
	Note: These sequences can vary depending on the desired fusion protein.</li>
	<li>Send complete amino acid sequence to a company for gene design/optimization for desired host species and synthesis. Subclone into a vector of choice using the BamHI-NotI site using a kit or by utilizing commercial services.</li>
</ol>
2. Making Agar Plates
Note: It is very important that all stocks of antibiotics, plates, buffers, etc. are stored properly (temperature and duration) and remain free of proteases (sterilized).
<ol>
	<li>Weigh out agar at 1.5 wt% and lysogeny broth (LB) at 20 g/L and place in a glass media bottle. A 500 ml bottle makes about 15 plates.</li>
	<li>Volumetrically add ultrapure water, mix well, and autoclave.</li>
	<li>Let bottle cool to the touch and add the appropriate antibiotics. Avoid premature solidification of agar; however, if bottle cools too much and solution begins to congeal, reheat in microwave. 
	Note: Our plasmid contains an ampicillin resistance gene. Therefore, all steps must contain ampicillin at 100 &#181;g/ml. The E. coli cloning K12 strain requires tetracycline (12.5 &#181;g/ml) antibiotic. BL21 bacteria cells do not require any additional antibiotic.</li>
	<li>Pour 25-30 ml of solution into 90 mm Petri dishes and allow time to dry.</li>
	<li>Label dish with appropriate antibiotics and store upside-down in 4 &#176;C, with Parafilm or in a resealable bag. 
	Note: Plates are good for about one month.</li>
</ol>
3. Cloning of the Biotin Tagged Plasmid
Note: Conditions are done aseptically.
<ol>
	<li>Spin down plasmid vector at 6,000 x g for 3 min in order to pellet, and add 10 &#956;l of sterilized ultrapure water.</li>
	<li>Remove 50 &#956;l of chemically competent E. coli high transformation efficiency cells from -80 &#176;C and immediately place on ice for 5-10 min.</li>
	<li>Add 1 &#956;l of vector solution at 0.5-1 ng/50 &#956;l cells to the 50 &#956;l E. coli cells and place the remaining plasmid in -80 &#176;C.</li>
	<li>Place the bacteria cells containing the vector on ice for 5 min.</li>
	<li>Heat shock the cell and vector mixture for 30-45 sec in 42 &#176;C water bath and place cells back on ice for 2 min.</li>
	<li>Add 250 &#956;l of super optimal broth with catabolite repression (SOC) medium (2% w/v bacto-tryptone, 0.5% w/v bacto-yeast extract, 8.56 mM NaCl, 2.5 mM KCl, 20 mM MgSO4, 20 mM glucose, ultrapure water; pH= 7.0) and shake at 250 rpm at 37 &#176;C for 30 min. 
	Note: SOC should not be autoclaved but be filter sterilized through a 0.22 &#181;m filter.</li>
	<li>Dry plates 10-15 min prior to plating cells.</li>
	<li>Pipette 25, 50, and 100 &#956;l of cell solution onto separate agar plates containing the ampicillin (100 &#181;g/ml) and tetracycline (12.5 &#181;g/ml) antibiotics. Use glass beads to distribute solution evenly over agar plates.</li>
	<li>Incubate plates upside-down at 37 &#176;C for 15-18 hr.</li>
	<li>Check for small individual bacteria colonies on plates and store upside-down at 4 &#176;C until further use (Figure 2A).
	<ol>
		<li>If no colonies are present:
		<ol>
			<li>Check the freshness and sterility of buffers and supplies.</li>
			<li>Increase the amount of plasmid added to E. coli K12 strain.</li>
		</ol>
		</li>
		<li>If large colonies are present or there is an overgrowth of colonies (Figure 2B and 2C), restreak a new agar plate using a sterile inoculation loop or plate cells at lower volume.</li>
	</ol>
	</li>
	<li>Inoculate 5 ml LB containing ampicillin (100 &#181;g/ml) and tetracycline (12.5 &#181;g/ml) antibiotics with a single colony of transformed E. coli cells. Grow-up cells overnight at 37 &#176;C at 250-300 rpm.</li>
	<li>The following day, use a plasmid isolation kit in order to isolate the target vector from the overnight grow-up and store purified plasmid at -80 &#176;C until further use.
	<ol>
		<li>Optional: Check purity and concentration of plasmid using absorbance reading obtained at 260 and 280 nm.</li>
	</ol>
	</li>
</ol>
4. Transformation of Plasmid into Expression Host
Note: Conditions are done aseptically.
<ol>
	<li>Repeat steps 3.2-3.10 for E. coli BL21 cells with the following changes: 
	Note: BL21 cells do not require any additional antibiotics, therefore only ampicillin is added to the agar plates for transformation.
	<ol>
		<li>Add 2-5 &#956;l of isolated plasmid at 0.5-2 ng/50 &#956;l cells from step 3.12 to 50 &#956;l E. coli expression host cells.</li>
		<li>Heat shock cells for 60-90 sec.</li>
		<li>Shake solution containing SOC medium at 250 rpm for 60 min instead of 30 min.</li>
	</ol>
	</li>
	<li>Pluck a single colony of transformed bacteria cells from the Petri dish with a sterile applicator stick and place in a test tube containing 5 ml LB broth with the appropriate concentration of ampicillin (100 &#181;g/ml).</li>
	<li>Place test tubes in shaker overnight at 37 &#176;C at 250 rpm.</li>
	<li>The following morning, take 200 &#956;l of the overnight grow-up and add it to 5 ml LB containing ampicillin at 100 &#181;g/ml.</li>
	<li>Freeze down the remaining cells with 250 &#956;l 50% glycerol (sterile) to 750 &#956;l culture and keep in -80 &#176;C. 
	Note: New test expression and scale-up procedures can be done by using a sterile applicator stick to obtain a scraping of frozen cells and inoculating LB with the appropriate antibiotics.</li>
	<li>Place test tube in shaker at 250-300 rpm at 37 &#176;C until optical density (OD) is measured between 0.7-0.8 at an absorbance of 600 nm.</li>
	<li>Induce cells with IPTG at a final concentration of 1 mM.</li>
	<li>Shake for an additional 4 hr at 37 &#176;C at 300 rpm. Alternatively cells can also be shaken overnight at 18 &#176;C at 250-300 rpm. This can produce a higher yield of plasmid depending on the target protein.</li>
	<li>Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis of Test Expression
	<ol>
		<li>Take 500 &#956;l of culture and place in a 1.5 ml tube. Spin down at 13-15 x g for 5 min. Pour off supernatant liquid and label "Soluble." Resuspend pellet with vortexing in 200 &#956;l of loading dye (50 &#956;l 2-mercaptoethanol, 950 &#956;l Laemmli sample buffer). 
		Note: Soluble bacteria pellets will be used to determine if the recombinant protein is in the cytoplasmic regions of the bacteria cells. Pellets can be stored in -80 &#176;C without loading dye until needed. A control bacteria culture that has not been transformed or induced can be treated as soluble as well for comparison.</li>
		<li>Take 1,000 &#956;l of culture and place in a 1.5 ml tube. Spin down at 13-15 x g for 5 min. Pour off supernatant liquid and label "Insoluble." 
		Note: Insoluble pellets will undergo denaturation procedures below in order to release proteins found in inclusion bodies of bacteria cells. Pellets can be stored in -80 &#176;C until needed. A control bacteria culture that has not been transformed or induced can be treated as insoluble as well for comparison.
		<ol>
			<li>Resuspend the insoluble pellet in 200 &#956;l Bugbuster and leave at room temperature for 30 min.</li>
			<li>Spin down sample again at 13-15 x g for 5 min and take 50 &#956;l of supernatant and add it to new "Insoluble" 1.5 ml tube.</li>
			<li>Then add 50 &#956;l of loading dye to this new sample.</li>
		</ol>
		</li>
		<li>Boil both soluble and insoluble samples for 5 min to denature proteins for SDS-PAGE analysis.</li>
		<li>Load each sample into electrophoresis gel with standard ladder.</li>
		<li>In order to visualize the protein samples use a staining reagent and follow the company&#8217;s protocol.</li>
		<li>Determine whether the protein expresses in the soluble and/or insoluble fractions by comparing these two lanes as well as comparing the lanes to the soluble and insoluble control lanes (see Note 4.9.1 and 4.9.2) on the SDS-PAGE gel (Figure 2). A dark band should appear around the molecular weight of the protein in the soluble or insoluble lanes. This will determine which isolation procedure to use in Protocol 6. 
		Note: If there is a band in both of these regions than either isolation used in Protocol 6 below can be performed, or the protein can be isolated both ways in order to determine which isolation method produces a higher yield of recombinant protein (Figure 3).</li>
		<li>If a 4 hr and overnight induction was conducted, determine which induction method has the highest protein production for the scale-up process (Figure 2).</li>
	</ol>
	</li>
</ol>
5. Scale-up Procedure and Main Culture
<ol>
	<li>Prepare growth media with 85.7 g of Terrific Broth and 28.8 ml of 50% glycerol in 1.8 L of ultrapure water and autoclave on liquid cycle for 1 hr.</li>
	<li>Start an overnight culture from frozen cell stock created in step 4.5. 
	Note: Conditions are done aseptically.
	<ol>
		<li>Mix 20 ml of LB and with a final concentration of ampicillin at 100 &#181;g/ml in a sterile 125 ml Erlenmeyer flask.</li>
		<li>Using a sterile applicator stick take a scrapping of transformed E. coli cells and drop applicator into the flask. Remove applicator stick and plug flask with foam stopper or cotton and cover with aluminum foil to maintain sterility.</li>
		<li>Shake overnight at 250 rpm at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Main Culture 
	Note: Conditions are done aseptically.
	<ol>
		<li>Pour overnight culture into 1.8 L of sterile growth media and add ampicillin at 100 &#181;g/ml and 6 to 8 drops of sterile antifoam 204 using a Pasteur pipette.</li>
		<li>Place cultures in a 37 &#176;C water bath with 0.2 mm filtered compressed air bubbling into cultures through aeration stones.</li>
		<li>Once the OD600nm reaches 0.7-0.8, induce with IPTG (1 mM) and allow induction to occur for either 4 hr at 37 &#176;C or overnight at 18 &#176;C depending on soluble/insoluble results from SDS-PAGE in step 4.9.</li>
	</ol>
	</li>
	<li>Collecting E. coli Cells
	<ol>
		<li>Spin down the cell culture in 1 L centrifuge bottles (2 bottles/culture) for 15 min at 14,000 x g at 4 &#176;C.</li>
		<li>Pour off supernatant and using a thin spatula, scoop the bacteria pellet into two 50 ml tubes. The cells can be pelleted again by centrifugation for a few minutes. 
		Note: Each 1.8 L culture will result in two bacteria pellets, one in each 50 ml centrifuge tube.</li>
		<li>Store pellets in -80 C until protein isolation.</li>
	</ol>
	</li>
</ol>
6. Isolation and Purification of Recombinant Protein
Note: If the protein is located in the soluble region based of SDS-PAGE Analysis from step 4.9 then "Native Isolation" will be used, but for insoluble proteins "Nonnative Isolation" procedures will be performed.
<ol>
	<li>Cell Lysis
	<ol>
		<li>Nonnative
		<ol>
			<li>Remove transformed E. coli pellet from -80 &#176;C freezer, and add 20 ml Lysis/Wash Buffer (Table 1) to each centrifuge tube.</li>
			<li>Vortex and shake the frozen pellet until it thaws and solubilizes into the buffer solution without any large chunks. Incubate on nutator overnight. The following morning, the pellet should now be a viscous slurry due to the denaturing of the protein from the buffer.</li>
			<li>Centrifuge the slurry at 20,500 x g at room temperature for 30 min. Transfer the supernatant to a fresh tube for isolation and discard the pellet.</li>
		</ol>
		</li>
		<li>Native
		<ol>
			<li>Obtain transformed E. coli pellet from -80 &#176;C freezer.</li>
			<li>Add Lysis Buffer (Table 1) to centrifuge tube to reach a final volume of 30 ml, and dislodge frozen pellet by vortexing and shaking rigorously. Place pellet on ice.</li>
			<li>Wash sonicator probe with 70% ethanol at 100% amplitude for 1-2 min. Then repeat with ultrapure water.</li>
			<li>Sonicate bacteria pellet while on ice for 5 min (total elapsed time of 10 min).
			<ol>
				<li>Set sonicator at 30% amplitude with pulse of 30 sec on/30 sec off.</li>
				<li>Bob centrifuge tube up and down during sonication interval in order to completely break up the cell pellet. At the end of the total elapsed time there should be no visible bacteria pellet leaving a stringy, viscous slurry.</li>
				<li>Clean the sonicator probe between different protein isolations as well as for storage using 70% ethanol and ultrapure water as described in step 6.1.2.3.</li>
			</ol>
			</li>
			<li>Centrifuge the slurry at 20,500 x g at 4 &#176;C for 30 min. Transfer the supernatant to a fresh tube for isolation and discard the pellet.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Ni-NTA Affinity Chromatography
	<ol>
		<li>Nonnative
		<ol>
			<li>Add 1 ml Ni2+-NTA resin solution to each centrifuge tube (2 ml resin total for a 1.8 L culture) and incubate at RT for at least 1 hr on nutator.</li>
			<li>Pour slurry into affinity column and allow solution to drip through stopcock valve completely into waste beaker.</li>
			<li>Perform 10 washes with 10 ml of Lysis/Wash Buffer for each wash.
			<ol>
				<li>For the first two washes, add the 10 ml to the centrifuge tube to remove additional resin and then pour into column. The additional washes can be added directly to column.</li>
				<li>After each wash, stir the resin with a glass stirring rod. Once the wash drips into the waste beaker, the next 10 ml can be added.</li>
			</ol>
			</li>
			<li>After wash completely drips through, close stopcock valve, remove waste beaker and replace with 50 ml centrifuge tube for protein collection.</li>
			<li>Add 15 ml of Elution Buffer (Table 1) and stir-up resin, allowing solution to sit for 5 min. Open stopcock valve and collect elution. Repeat again with an additional 15 ml.</li>
		</ol>
		</li>
		<li>Native
		<ol>
			<li>Follow the Nonnative Affinity Chromatography above (Steps 6.2.1.1-6.2.1.4) except adjust the following parameters:
			<ol>
				<li>Incubate Ni2+-NTA resin solution at 4 &#176;C for at least 1 hr on nutator.</li>
				<li>Use the appropriate Wash Buffer (Table 1).</li>
			</ol>
			</li>
			<li>Add 5 ml of Elution Buffer (Table 1) and incubate with resin for 5 min.
			<ol>
				<li>Open stopcock valve and collect elution until most of the solution has dripped through.</li>
				<li>Add 90 &#956;l/well of Bradford reagent to clear 96-well plate. After each elution allow 10 &#956;l of solution to drip from column into well containing 90 &#956;l Bradford reagent.</li>
				<li>Continue adding 5 ml Elution Buffer at a time until Bradford assay no longer detects protein (color goes from dark blue to light blue to clear). This usually takes 4-5 elution volumes.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Dialysis/Renaturation
	<ol>
		<li>Make nonnative (renaturation) and native dialysis buffers (Table 1) and store in 4 &#176;C. 
		Note: The pH of the dialysis buffers are determined by the target protein&#8217;s isoelectric point (pI). As a rule of thumb proteins should be buffered at least one full point above or below their pI values.</li>
		<li>Transfer native and nonnative elutions to dialysis tubing with appropriate molecular weight cutoff (25,000 MWCO for NGF and 50,000 MWCO for Sema3A). Place each protein sample in respective dialysis buffer 1 for 4 hr at 4 &#176;C and then replace with the respective buffer 2 overnight at 4 &#176;C. 
		Note: Protein needs to be dialyzed in each buffer for at least 4 hr for proper refolding and buffer exchange to take place.</li>
		<li>Concentrate the dialyzed protein elutions to less than 5 ml using centrifuge spin concentrators. 
		Note: Proteins can further be purified by using fast protein liquid chromatography (FPLC) and concentrated again.</li>
	</ol>
	</li>
	<li>Determine protein concentration (mg/ml) by freeze drying a 10-50 ml sample in a preweighed microcentrifuge tube.
	<ol>
		<li>Optional: determine the extinction coefficient, &#949;, so that concentration of protein can be determined in future isolations by measuring the absorbance of a sample at 280 nm using Beer-Lambert&#8217;s Law: c=A280nm/&#949;l, where l is the path length.</li>
	</ol>
	</li>
</ol>
7. Biotinylation of Purified Protein
<ol>
	<li>Dialyze proteins in 10,000 MWCO dialysis cassettes against 10 mM Tris (pH=8.0), changing the buffer every 4 hr with a total of 6 changes.</li>
	<li>Transfer the recombinant proteins (now in 10 mM Tris buffer) to 2 ml micro-centrifuge tubes for biotinylation.</li>
	<li>Biotinylate proteins using a biotin protein ligase kit.</li>
	<li>Dialyze proteins against PBS (pH=7.4) using 10,000 MWCO dialysis cassettes with 3 buffer changes a day for 2 days.</li>
	<li>Determine the percent biotinylation of proteins using a quantitation kit.</li>
	<li>Determine the concentration again as described in 6.4 and store at 4 &#176;C or aliquot and store at -20 &#176;C for long term storage.</li>
</ol>

The authors have nothing to disclose.

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...

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 somatostatin sequence was one of the first engineered polypeptides created using E. coli1. Soon after in 1979, insulin was cloned in plasmid pBR322, transformed, expressed, and purified2. Since then, recombinant proteins have expanded their influence to multiple fields of research such as biomaterials, drug delivery, tissue engineering, biopharmaceuticals, farming, industrial enzymes, biofuels, etc. (for reviews see references3-8). This is largely due to the versatility that the technique offers via the addition of application specific chemical moieties or protein sequences for purposes including, but not limited to, target protein identification, stabilization and purification.

Via recombinant DNA technology, recombinant proteins can be expressed in a variety of eukaryotic and prokaryotic host systems including mammalian, plant, insect, yeast, fungus, or bacteria. Each host offers different advantages and typically the best system is determined based on protein function, yield, stability, overall cost, and scalability. Bacteria cells often lack the post-translational modification mechanisms that eukaryotic hosts provide (i.e.&#160;glycosylation, disulfide bridging, etc.)5. As a result, mammalian and insect systems usually result in better compatibility and expression of eukaryotic proteins; however these hosts are typically more expensive and time-consuming9. Therefore, E. coli is the favored host for our expression system because cells expand rapidly in inexpensive growth conditions and the genetic expression mechanisms are well understood5,9. Additionally, this system is easy to scale-up for production purposes and results in functional proteins despite the lack of post-translational modifications10. The E. coli K12 strain is chosen in this protocol for cloning because this strain offers excellent plasmid yields based on high transformation efficiencies. Additionally, an E. coli BL21 strain is utilized for expression because this host strain contains the T7 RNA polymerase gene which provides controlled protein expression and stability11.
After host selection, further care must be taken in selecting the ideal expression vector to facilitate selected and controlled protein expression. Synthesizing recombinant proteins begins with a target DNA sequence that is cloned under the direction of bacteriophage T7 transcription and translation signals, and expression is induced in host cells containing chromosomal copies of the T7 RNA polymerase gene12. These vectors, derived from plasmid vector pBR322 (for review see reference13), are tightly controlled by the T7 promoter initially developed by Studier and colleagues14 and provide additional control through inclusion of the lac operator and lac repressor (lac1)15,16. For recombinant protein engineering, this expression system offers the ability to tailor a specific amino acid sequence of a desired protein by inserting different target DNA sequences or to create fusion proteins made up of combined domains from single proteins. Additionally, some vector series include peptide tag modifications to be placed on the N or C terminus. For our design purposes, a histidine (His) tag was added to the DNA target sequence for purification and a 15 amino acid biotinylatable sequence was included for biotinylation17,18. In this protocol a plasmid containing an ampicillin resistance gene, was chosen to carry our biotinylatable fusion protein sequences. Expression is controlled in this vector via the T7 lac promoter and is easily induced with isopropyl &#946;-D-1-thiogalactopyranoside (IPTG).
Test expressions (small-scale cultures) are used to determine the presence and solubility of the target protein, which can be expressed in either a soluble or insoluble form to formulate purification procedures. A soluble protein expressed within the bacteria cell will undergo spontaneous folding to maintain its native structure19. Typically the native structure is thermodynamically favorable. In many cases the metabolic activity of the host is not conducive to the target protein, placing stress on the system that leads to insoluble protein production and the formation of inclusion bodies composed of insoluble protein aggregates. Thus the target protein denatures, rendering them generally biologically inactive20. Both test expressions are scaled-up, and isolation procedures are determined by the solubility of the target protein. An additional renaturation or refolding step is required for insoluble proteins. The resulting recombinant proteins can be further purified using size exclusion chromatography.
In house recombinant protein production offers cost advantages over commercial products since milligrams of target protein can be isolated per liter of main culture. Most of the required equipment is available in a typical biological or chemical laboratory. Protein engineering allows for creation of custom fusion proteins with added functionalities which are not always commercially available. Figure 1 depicts the main procedures involved in engineering recombinant proteins. With this expression system we have created many biotinylatable proteins, such as interferon-gamma, platelet-derived growth factor, and bone-morphogenetic protein21-23, but we will focus on two proteins that we designed for axon guidance, NGF (29 kDa) and Sema3A (91 kDa)10 (for review see reference24). Biotinylation is a common technique for identification, immobilization and isolation of labeled proteins utilizing the well-known biotin-streptavidin interaction25-27. Biophysical probes28,29, biosensors30, and quantum dots31 are some examples of systems that utilize the high affinity of biotin-streptavidin conjugation with a Kd on the order of 10-15 M&#160;27. The E. coli biotin ligase, BirA, aids in the covalent attachment of biotin to the lysine side chain found within the biotin tagged sequence18,32. Tethering biotin to materials and biomolecules has produced sustained delivery of growth factors to cell for multiple tissue engineering applications21,33-35. Therefore, engineering these custom-designed biotinylatable proteins is a powerful tool that can transcend multiple research interests.

<table border="0" cellpadding="0" cellspacing="0" width="849">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">1,4-dithio--DL-threitol, DTT, 99.5%</td>
			<td style="width:191px;">Chem-Impex International</td>
			<td style="width:121px;">127</td>
			<td style="width:239px;">100 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">2-Hydroxyethylmercaptan &#946;-Mercaptoethanol</td>
			<td>Chem-Impex International</td>
			<td>642</td>
			<td>250 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid, glacial</td>
			<td>EMD</td>
			<td>AX0073-9</td>
			<td>2.5 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agar</td>
			<td>Bioshop</td>
			<td>AGR001.500</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ampicillin sodium salt&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A9518</td>
			<td>25 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antifoam 204</td>
			<td>Sigma-Aldrich</td>
			<td>A6426</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Barstar-NGF pET-21a(+)</td>
			<td>GenScript USA Inc.</td>
			<td></td>
			<td>4 &#181;g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BL21(DE3) Competent Cells</td>
			<td>Novagen</td>
			<td>69450</td>
			<td>1 ml; Expression Host</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bradford reagent</td>
			<td>Sigma-Aldrich</td>
			<td>B6916</td>
			<td>500 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BugBuster</td>
			<td>Novagen</td>
			<td>70922-3</td>
			<td>100 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gel filtration standard</td>
			<td>Bio-Rad</td>
			<td>151-1901</td>
			<td>6 vials</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>Bioshop</td>
			<td>GLY001.1</td>
			<td>1 L</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:299px;">Guanidine hydrodioride amioformamidine hydrochloride</td>
			<td>Chem-Impex International</td>
			<td>152</td>
			<td>1 kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">His-Pur Ni-NTA Resin</td>
			<td>Thermo Scientific</td>
			<td>88222</td>
			<td>100 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hydrochloric acid</td>
			<td>EMD&#160;</td>
			<td>HX0603-3</td>
			<td>2.5 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imidazole&#160;</td>
			<td>Chem-Impex International</td>
			<td>418</td>
			<td>250 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IPTG</td>
			<td>Chem-Impex International</td>
			<td>194</td>
			<td>100 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laemmli sample buffer</td>
			<td>Bio-Rad</td>
			<td>161-0737</td>
			<td>30 ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:299px;">Lauryl sulfate sodium salt, Sodium dodecyl surface</td>
			<td>Chem-Impex International</td>
			<td>270</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LB Broth&#160;&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td>1 kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NovaBlue Competent Cells</td>
			<td>Novagen</td>
			<td>69825</td>
			<td>1 ml; Cloning Host</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P5368-10PAK</td>
			<td>10 pack</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium Chloride</td>
			<td>Chem-Impex International</td>
			<td>01247</td>
			<td>1 kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sema3A-pET-21a(+)</td>
			<td>GenScript USA Inc.</td>
			<td></td>
			<td>4 &#181;g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SimplyBlue SafeStain</td>
			<td>Invitrogen</td>
			<td>LC6060</td>
			<td>1 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S5886-1KG</td>
			<td>1 kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S318-500</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium phosphate diabasic</td>
			<td>Sigma-Aldrich</td>
			<td>S5136-500G</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>S5011</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Terrific Broth</td>
			<td>Bioshop</td>
			<td>TER409.5</td>
			<td>5 kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tetracycline hydrochloride</td>
			<td>Chem-Impex International</td>
			<td>667</td>
			<td>25 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris/Glycine/SDS Buffer, 10X</td>
			<td>Bio-Rad</td>
			<td>1610732</td>
			<td>1 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trizma Base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td>1 kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tryptone, pancreatic</td>
			<td>EMD</td>
			<td>1.07213.1000</td>
			<td>1 kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Yeast extract, granulated</td>
			<td>EMD</td>
			<td>1.03753.0500</td>
			<td>500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:299px;">Name of Kits/ Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:121px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">&#160;&#196;KTApurifier10</td>
			<td style="width:191px;">GE Healthcare</td>
			<td style="width:121px;">28-4062-64&#160;</td>
			<td>Includes kits and accessories</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Benchtop Orbital Shaker</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:121px;">SHKE4000</td>
			<td>MAXQ 4000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">BirA500</td>
			<td style="width:191px;">Avidity</td>
			<td style="width:121px;">BirA500</td>
			<td style="width:239px;">Enzyme comes with reaction buffers and biotin solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Dialysis Casette</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:121px;">66380</td>
			<td>Slide-A-Lyzer (Extra Strength)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Dialysis Tubing</td>
			<td style="width:191px;">Spectrum Laboratories&#160;</td>
			<td style="width:121px;">132127, 132129</td>
			<td style="width:239px;">MWCO: 25,000 and 50,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Flow Diversion Valve FV-923</td>
			<td style="width:191px;">GE Healthcare</td>
			<td style="width:121px;">11-0011-70</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">FluoReporter Biotin Quantification Assay Kit</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:121px;">1094598</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Frac-950 Tube Racks, Rack C</td>
			<td style="width:191px;">GE Healthcare</td>
			<td style="width:121px;">18-6083-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Fraction Collector Frac-950</td>
			<td style="width:191px;">GE Healthcare</td>
			<td style="width:121px;">18-6083-00</td>
			<td>Includes kits and accessories</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Heated/Refrigerated Circulator&#160;</td>
			<td style="width:191px;">VWR</td>
			<td style="width:121px;">13271-102</td>
			<td>Model 1156D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Heating Oven FD Series</td>
			<td style="width:191px;">Binder</td>
			<td style="width:121px;"></td>
			<td>Model FD 115</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">HiLoad 16/60 Superdex 200 pg</td>
			<td style="width:191px;">GE Healthcare</td>
			<td style="width:121px;">17-1069-01</td>
			<td style="width:239px;">Discontinued--Replacement Product: HiLoad 16/600 Superdex 200 pg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">J-26 XPI Avanti Centrifuge</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:121px;">393126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">JA 25.50 Rotor</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:121px;">363055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">JLA 8.1 Rotor</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:121px;">969329</td>
			<td>Includes 1 L polyporpylene bottles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">JS 5.3 Rotor</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:121px;">368690</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Laminar Flow Hood</td>
			<td style="width:191px;">Themo Scientific</td>
			<td style="width:121px;">1849</td>
			<td style="width:239px;">Forma 1800 Series Clean Bench</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Microplate Reader</td>
			<td style="width:191px;">TECAN</td>
			<td style="width:121px;"></td>
			<td>infinite M200</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Mini-PROTEAN Tetra Cell</td>
			<td style="width:191px;">Bio-Rad</td>
			<td style="width:121px;">165-8004</td>
			<td style="width:239px;">4-gel vertical electrophoresis system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Mini-PROTEAN TGX Precast Gels</td>
			<td style="width:191px;">Bio-Rad</td>
			<td style="width:121px;">456-9036</td>
			<td style="width:239px;">Any kDa, 15-well comb</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Ni-NTA Column</td>
			<td>Bio-Rad</td>
			<td>737-2512</td>
			<td>49 ml&#160;volume ECONO-Column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Plasmid Miniprep Kit</td>
			<td style="width:191px;">OMEGA bio-tek</td>
			<td style="width:121px;">D6943-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">PowerPac HC Power Supply</td>
			<td>Bio-Rad</td>
			<td style="width:121px;">164-5052</td>
			<td>250 V, 3 A, 300 W</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Round Bottom Polypropylene Copolymer Tubes</td>
			<td style="width:191px;">VWR</td>
			<td style="width:121px;">3119-0050</td>
			<td>50 ml&#160;tubes for JA 25.50 rotor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Spin-X UF Concentrators</td>
			<td style="width:191px;">Corning</td>
			<td style="width:121px;">431488, 431483</td>
			<td>&#160;20 and 6 ml; MWCO: 10,000 Da</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Subcloning Service</td>
			<td style="width:191px;">GenScript USA Inc.</td>
			<td style="width:121px;"></td>
			<td>Protein Services</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Ultrasonic Processor&#160;</td>
			<td style="width:191px;">Cole-Parmer</td>
			<td style="width:121px;">18910445A</td>
			<td>Model CV18</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Vortex-Genie 2</td>
			<td style="width:191px;">Scientific Industries</td>
			<td>SI-0236</td>
			<td>Model G560</td>
		</tr>
	</tbody>
</table>

expression, isolation, and purification of soluble and insoluble biotinylated proteins for nerve tissue regeneration

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&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

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 &#176;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...

Watch this Scientific Journal Video about Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration at JoVE.com

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

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.

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

expression-isolation-purification-soluble-insoluble-biotinylated

Research

JoVE Journal

Bioengineering

33.5K ビュー.  University of Akron.  biotinylatable融合タンパク質を開発することは、研究の様々な分野で多くの潜在的な用途がある。組換えタンパク質工学は、カスタム設計されたタンパク質を高収率で提供する、費用対効果のある単純な手順である。 

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

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the wet spinning method, gelatin fibers are produced by smooth extrusion in a suitable coagulation medium. To increase the functional surface of these gelatin fibers and their ability to mimic the features of tissues, gelatin can be molded into a tube form by referring to this concept. Examined by in vitro and in vivo tests, the gelatin tubes demonstrate a great potential for application in tissue engineering. Acting as a suitable filling gap material, gelatin tubes can be used to substitute the tissue in the damaged area (e.g., in the nervous or cardiovascular system), as well as to promote regeneration by providing a direct replacement of stem cells and neural circuitry. This protocol provides a detailed procedure for creating a biomaterial based on a natural polymer, and its implementation is expected to greatly benefit the development of correlative natural polymers, which help to realize tissue regeneration strategies.

We developed and describe a protocol based on the wet spinning concept, for the construction of gelatin-based biomaterials used for the application of tissue engineering.

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the ...