Name: oaaep1-mediated enzymatic synthesis and immobilization of polymerized protein for single-molecule force spectroscopy
Uploaded: 2020-02-05T14:30:00
Description: Watch this Scientific Journal Video about OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy at JoVE.com

This work was supported by the National Natural Science Foundation of China (Grant No. 21771103, 21977047), Natural Science Foundation of Jiangsu Province (Grant No. BK20160639) and Shuangchuang Program of Jiangsu Province.

1. Protein production
<ol>
	<li>Gene clone
	<ol>
		<li>Purchase genes coding for the protein of interest (POI): Ubiquitin, Rubredoxin (RD)51, the cellulose-binding module (CBM), dockerin-X domain (XDoc) and cohesion from Ruminococcus flavefacience, tobacco etch virus (TEV) protease, elastin-like polypeptides (ELPs).</li>
		<li>Perform polymerase chain reaction and use three-restriction digestion enzyme system BamHI-BglII-KpnI for recombining t...

We have described a protocol for enzymatic biosynthesis and immobilization of polyprotein and verified the polyprotein design by AFM-based SMFS. This methodology provides a novel approach to building the protein-polymer in a designed sequence, which complements previous methods for polyprotein engineering and immobilization4,6,52,53,54,<sup class="x...

Compared with synthetic polymers, natural multi-domain proteins have a uniform structure with a well-controlled number and type of subdomains1. This feature usually leads to improved biological function and stability2,3. Many approaches, such as cysteine-based disulfide bond coupling and recombinant DNA technology, have been developed for building such a polymerized protein with multiple domains4,5,6,7. However, the former method always results in a random and uncontrolled sequence, and the latter one leads to other problems, including the difficulty for the overexpression of toxic and large-size proteins and the purification of complex protein with cofactor and other delicate enzymes.
To meet this challenge, we develop an enzymatic method that conjugates protein monomer together for polymer/polyprotein in a stepwise fashion using a protein ligase OaAEP1 combined with a protease TEV8,9. OaAEP1 is a strict and efficient endopeptidase. Two proteins can be linked covalently as Asn-Gly-Leu sequence (NGL) through two termini by OaAEP1 in less than 30 min if the N-terminus is Gly-Leu residues(GL) and the other of which the C-terminus is NGL residues10. However, the use of OaAEP1 only to link protein monomer leads to a protein polymer with an uncontrolled sequence like the cysteine-based coupling method. Therefore, we design the N-terminus of the protein unit with a removable TEV protease site plus a leucine residue as ENLYFQ/G-L-POI. Before the TEV cleavage, the N-terminal would not participate in OaAEP1 ligation. And then the GL residues at N-terminus, which are compatible with further OaAEP1 ligation, is exposed after the TEV cleavage. Thus, we have achieved a sequential enzymatic biosynthesis method of polyprotein with a relatively well-controlled sequence.
Here, our stepwise enzymatic synthesis method can be used in polyprotein sample preparation, including sequence-controlled and uncontrolled, and protein immobilization for single-molecule studies as well, especially for the complex system such as metalloprotein.
Moreover, AFM-based SMFS experiments allow us to confirm the protein polymer construction and stability at the single-molecule level. Single-molecule force spectroscopy, including AFM, optical tweezer and magnetic tweezer, is a general tool in nanotechnology to manipulate biomolecule mechanically and measure their stability11,12,13,14,15,16,17,18,19,20. Single-molecule AFM has been widely used in the study of protein (un)folding21,22,23,24,25, the strength measurement of receptor-ligand interaction26,27,28,29,30,31,32,33,34,35, inorganic chemical bond20,36,37,38,39,40,41,42,43 and metal-ligand bond in metalloprotein44,45,46,47,48,49,50. Here, single-molecule AFM is used to verify the synthesized polyprotein sequence based on the corresponding protein unfolding signal.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>iron (III) chloride hexahydrate</td>
			<td>Energy chemical</td>
			<td></td>
			<td>99%</td>
		</tr>
		<tr>
			<td>Zinc chloride</td>
			<td>Alfa Aesar</td>
			<td></td>
			<td>100.00%</td>
		</tr>
		<tr>
			<td>calcium chloride hydrate</td>
			<td>Alfa Aesar</td>
			<td></td>
			<td>99.9965% crystalline aggregate</td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid</td>
			<td>Sigma Life Science</td>
			<td></td>
			<td>Bio Xtra, &#8805;99.0%, crystalline</td>
		</tr>
		<tr>
			<td>(3-Aminopropyl) triethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>&#8805;99%</td>
		</tr>
		<tr>
			<td>sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>90%</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Macklin</td>
			<td></td>
			<td>99%</td>
		</tr>
		<tr>
			<td>5,5&#39;-dithiobis(2-nitrobenzoic acid)</td>
			<td>Alfa Aesar</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Genes</td>
			<td>Genscript</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanowizard 4 AFM</td>
			<td>JPK Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MLCT cantilever</td>
			<td>Bruker Corp</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mono Q 5/50 GL</td>
			<td>GE Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AKTA FPLC system</td>
			<td>GE Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>Sail Brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop 2000</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti JXN-30 Centrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Image System</td>
			<td>Tanon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

oaaep1-mediated enzymatic synthesis and immobilization of polymerized protein for single-molecule force spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

The NGL residues introduced between adjacent proteins by OaAEP1 ligation will not affect protein monomer stability in the polymer as the unfolding force (&#60;Fu&#62;), and contour length increment (&#60;&#916;Lc&#62;) is comparable with the previous study (Figure 1). The purification result of the rubredoxin protein is shown in Figure 2. To prove the protein after TEV cleavage is compatible with the following OaAEP1 ligation to construct p...

Watch this Scientific Journal Video about OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy at JoVE.com

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

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