Name: isolation and characterization of chimeric human fc-expressing proteins using protein a membrane adsorbers and a streamlined workflow
Uploaded: 2014-01-08T14:15:00
Description: Watch this Scientific Journal Video about Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow at JoVE.com

The authors wish to thank Mr. Luis F. Delgadillo (Department of Chemical and Biomolecular Engineering, Ohio University), Mr. Matthew H. Williams (Department of Chemical and Biomolecular Engineering, Ohio University), and Dr. Filiberto Cedeno-Laurent (Brigham and Women&#8217;s Hospital and Harvard Medical School) for expert technical assistance.&#160;The authors also wish to thank the Winter 2011 CHE 404/BME 504 and Fall 2012 CHE 4830/BME 5830 students (Department of Chemical and Biomolecular Engineering and Biomedical Engineering Program, Ohio University) for technical advice and discussion. This work was supported by National Science Foundation (NSF) Major Research Instrumentation grant CBET-1039869 (MMB), NSF CBET-1106118 (MMB), Dermatology Foundation Research Grant A050422 (SRB), National Institutes of Health (NIH) NCI grant 1R15CA161830-01 (MMB), NIH Kirschstein-NRSA Postdoctoral Fellowship F32CA144219-01A1 (SRB), NIH/NCI R01CA173610 (CJD),&#160;and NIH NCCAM grant R01AT004628 (CJD).

1. Verify the Affinity for Protein A
<ol>
	<li>Verify that the Fc-expressing protein (recombinant fusion protein or IgG antibody, heretofore referred to as "protein") has acceptable affinity for protein A14-17 prior to using the protein A membrane adsorber, and test for the presence of the protein in the stock solution (e.g. cell culture supernatant clarified by centrifugation at 1,000 x g for 5 min to pellet suspended cells). Use flow cytometry, Western blotting, ELISA, etc. to detect prot...

<ol>
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The protocol described herein was developed to rapidly isolate and characterize a chimeric fusion protein expressing human Fc (Gal-1hFc), without significantly compromising product recovery or inflating cost.&#160;The key components of the streamlined workflow are a protein A membrane adsorber for isolation, and semidry transfer and vacuum-assisted immunoblotting for characterization by Western blotting.
The dramatic decrease in processing time for isolation and characterization of Gal-1hFc fr...

Isolation of antibodies and recombinant proteins expressing immunoglobulin G (IgG) Fc fragments from cell culture supernatants and dilute lysed cell solutions can be accomplished using protein A affinity chromatography. In basic science and engineering laboratories and industry, columns packed with porous protein A-coated agarose, glass, or polymeric beads are most commonly used for affinity chromatography, despite high financial costs and long processing times1-3. It is well-appreciated that affinity chromatography represents the largest expense and processing bottleneck in both lab and industrial settings2,4. As a result, numerous improvements have been developed to decrease cost and processing time without sacrificing overall product recovery from the process train1,2,5-7. Particularly promising for the isolation of antibodies and other Fc-expressing proteins is affinity chromatography using a protein A membrane adsorber2,5,7-10. Whereas Fc capture in column chromatography is diffusion-limited (i.e.&#160;the Fc-expressing protein must diffuse into and through internal pores to reach the majority of the protein A) with high pressure drop across the column, mass transport in membrane adsorbers is driven by bulk convection, resulting in avoidance of diffusion limitations8,9,11,12. In addition, pressure drop across the membrane adsorber is low, thereby permitting faster perfusion flow rates compared to bead-packed columns8,9,11,12. Thus, for laboratory scale isolation, membrane chromatography is predicted to reduce isolation time by several hours and increase product capture compared to column chromatography. Furthermore, mathematical models for IgG adsorption in membrane adsorbers have been proposed8,9,11,12, thus allowing end-users to predict performance in the lab.
Another bottleneck in basic science and engineering laboratories is the characterization of the Fc-expressing protein. However, selection of appropriate methods to complete characterization assays, such Western blots, can greatly reduce time spent on product testing.&#160;For instance, semidry transfer in a discontinuous buffer system can accomplish electrophoretic transfer of proteins from an SDS-PAGE gel to a polyvinyl difluoridine (PVDF) membrane in a matter of minutes, as opposed to 1-2 hr in tank (wet) transfer in a continuous buffer system13.
A rapid, inexpensive, and effective protocol to isolate and characterize a chimeric fusion protein expressing an Fc fragment of human IgG is described herein. Most equipment is readily available in standard basic biomedical science, biology, chemistry, and (bio)chemical engineering labs, and all reagents are commercially available.&#160;Although representative results were generated from the isolation and characterization of a murine galectin-1/human Fc chimeric fusion protein (Gal-1hFc), the streamlined protocol can be applied to the isolation of other molecules that possess an Fc fragment, such as antibodies, Fc fragments themselves, or other Fc fusion proteins.&#160;Our improvements dramatically decreased processing time (as few as 4 hr but more typically ~9 hr, compared to 20+ work hours) while achieving similar or improved product recovery compared to standard methods.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Protein A membrane adsorber (Sartobind Protein A 2 ml)&#160;</td>
			<td style="width:191px;">Sartorius-Stedim</td>
			<td style="width:147px;">93PRAP06HB-12--A</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Amicon Ultra centrifugal filter, 10 kDa, 4 ml</td>
			<td style="width:191px;">Millipore</td>
			<td>UFC801008</td>
			<td>Use 15 ml volume if needed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Sterile vacuum filter unit, 0.22 &#181;m, 500 ml</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">10 ml Syringes with Luer Lock tips</td>
			<td>BD</td>
			<td>301604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">30 ml Syringes with&#160; Luer Lock tips</td>
			<td>BD</td>
			<td>309650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Tygon lab tubing (1/16 in x 1/8 in x 50 ft)</td>
			<td>Cole Parmer</td>
			<td>WU-95903-16</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Slide-A-Lyzer MINI Dialysis Devices (10K MWCO)</td>
			<td>Thermo Scientific</td>
			<td>69576</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Snap i.d. antibody collection tray</td>
			<td>EMD Millipore</td>
			<td>WBAVDABTR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Snap i.d. single well blot holder</td>
			<td>EMD Millipore</td>
			<td>WBAVDBH01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Elution buffer</td>
			<td>Thermo Scientific</td>
			<td>21004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Tris, ultra pure</td>
			<td>Fisher Scientific</td>
			<td>819623</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">DPBS</td>
			<td>Thermo Scientific&#160;</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:313px;">DPBS with Ca2+/Mg2+</td>
			<td>Life Technologies</td>
			<td>14080-055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">BSA</td>
			<td>Sigma</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Human Fc</td>
			<td>Bethyl Research Laboratories</td>
			<td>&#160;P80-104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Anti-human Fc-APC</td>
			<td>Jackson Immunoresearch</td>
			<td>109136170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Anti-human Fc-AP</td>
			<td>Bio-Rad</td>
			<td>170-5018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Alkaline phosphatase substrate</td>
			<td>Promega</td>
			<td style="width:147px;">S3841</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Flow cytometry tubes (5 ml polystyrene or polypropylene)</td>
			<td>BD</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;"></td>
			<td style="width:191px;"></td>
			<td style="width:147px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Name of Material/ Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>55-2226</td>
			<td style="width:173px;">A peristaltic pump is also acceptable</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Protein gel electrophoresis system&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>552BR094876</td>
			<td style="width:173px;">Any gel electrophoresis system is acceptable, but mini gel systems run fastest</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;">Semidry blotter&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td style="width:147px;">690BR006163</td>
			<td style="width:173px;">Any transfer system is acceptable, but discontinuos transfer systems perform electrophoretic transfer fastest</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:313px;">SNAP i.d. vacuum-assisted protein detection system</td>
			<td style="width:191px;">EMD Millipore</td>
			<td>WBAVDBASE</td>
			<td style="width:173px;">An upgraded model has replaced this specific instrument, but either should work just as well</td>
		</tr>
	</tbody>
</table>

isolation and characterization of chimeric human fc-expressing proteins using protein a membrane adsorbers and a streamlined workflow

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography.&#160;While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process.&#160;Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.).&#160;Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment.&#160;To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available.&#160;To demonstrate this protocol, representative results are presented in which chimeric&#160;murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber.&#160;Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods.

The streamlined protocol for isolation and characterization of Fc-expressing proteins is routinely used to process chimeric murine galectin-1 fused to human Fc (Gal-1hFc) from dilute cell culture supernatants. The flowchart in Figure 2 illustrates the workflow and time for each step in the protocol. For a typical batch of 300 ml of supernatant, the total processing time is approximately 9 hr when the optional flow cytometry testing of elution fractions is performed. If all flow cytometry analysis is omit...

Watch this Scientific Journal Video about Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow at JoVE.com

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Compared with traditional affinity chromatography using protein A agarose bead-packed columns, protein A membrane adsorbers can significantly speed laboratory-scale isolation of antibodies and other Fc fragment-expressing proteins.&#160;Appropriate analysis and quantification methods can further accelerate protein processing, allowing isolation/characterization to be completed in one workday, instead of 20+ work hours.

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using ...

Research

JoVE Journal

Bioengineering

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

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 ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...