<meta charset="utf8"></head><body>Avaliando a estabilidade sérica de HsADA1: uma abordagem baseada em microplacas

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<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adenosine&#160;</td>
			<td>Sigma Aldrich</td>
			<td>A9251-25G</td>
			<td>25 g</td>
		</tr>
		<tr>
			<td>BioTek Synergy HT Microplate Reader</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf LoBind Microcentrifuge Tubes: Protein&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>13-698-795</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific&#160;</td>
			<td>G33-4</td>
			<td>4 L</td>
		</tr>
		<tr>
			<td>HsKYNase66-W102H-T333N</td>
			<td>In-house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human Serum, Pooled</td>
			<td>MP Biomedicals</td>
			<td>92931149</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Hydroxy-kynurenine</td>
			<td>Cayman Chemicals</td>
			<td>27778</td>
			<td></td>
		</tr>
		<tr>
			<td>Inosine&#160;</td>
			<td>TCI</td>
			<td>I0037</td>
			<td>25 g</td>
		</tr>
		<tr>
			<td>PBS, 1x&#160; pH 7.4+/- 0.1</td>
			<td>Corning</td>
			<td>21-040-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridoxal 5-phosphate monohydrate, 99%&#160;</td>
			<td>Thermo Scientifc</td>
			<td>228170010</td>
			<td>1 g</td>
		</tr>
		<tr>
			<td>UV-STAR MICROPLATE, 96 WELL, COC, F-BOTTOM</td>
			<td>Greiner Bio</td>
			<td>&#160;655801</td>
			<td></td>
		</tr>
		<tr>
			<td>Wildtype Human Adenosine Deaminase 1</td>
			<td>In-house</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

determining the serum stability of human adenosine deaminase 1 enzyme

The following method allows a user to quantitatively assess an enzyme&#39;s ability to retain its activity when exposed to conditions that mimic what it will encounter following intravenous injection. The in vitro method mimics such in vivo conditions and consists of the incubation of the enzyme in pooled human serum at 37 &#176;C and time-coursed analyses of retention of enzyme activity. We refer to an enzyme&#39;s ability to retain activity in these conditions as its serum stability, and the analysis method for enzyme activity takes advantage of differences in absorbance between an enzyme&#39;s substrate and the resulting product. The concept of serum stability is not just enzyme-specific and has been applied to several other treatment modalities as well. For example, the serum stability of RNA aptamers targeting COVID spike proteins has previously been assessed by monitoring their degradation post-incubation with fetal calf serum1. Antibacterial peptides have also been assessed for their ability to suppress bacterial growth post-incubation with pooled human serum2.
HsADA1 is an enzyme that catalyzes the conversion of adenosine or 2-deoxyadenosine into inosine or 2-deoxyinosine, respectively3. Adenosine has an absorption peak of 260 nm, while inosine absorbs strongest at 250 nm4. This shift in absorption peaks can be detected on a microplate reader by a decrease in absorption intensity at 260 nm when HsADA1 is added to adenosine. The HsADA1 enzyme has important implications in the human body, and its deficiency causes a severe combined immunodeficiency (ADA-SCID)5. The bovine homolog of HsADA1, BtADA1, can be utilized as an enzyme replacement therapeutic for the treatment of ADA-SCID, and we have previously shown that wildtype HsADA1 loses its activity when incubated with pooled human serum, potentially hindering its use as a therapeutic6. Therefore, we selected the wildtype HsADA1 enzyme to demonstrate a procedure to determine an enzyme&#39;s serum stability. A detailed purification method for HsADA1 has been described previously6.
In the following protocol (as detailed in Figure 1), we demonstrate how to co-incubate wildtype HsADA1 in pooled human serum at 37&#160;&#176;C. During this time, test samples are taken at defined time points and flash-frozen for future analysis. Once all samples have been taken, a microplate assay is run whereby each sample is combined with the substrate, with the resulting absorbance decrease being a correlation for the retained activity of the enzyme. Representative results illustrating the serum stability of HsADA1 are shown, and because this metric may be relevant to determining the potential therapeutic value of other enzymes, we also discuss considerations to adapt this protocol to an engineered Homo sapiens kynureninase enzyme (HsKYNase) and any enzymes more generally. HsKYNase is an enzyme involved in the metabolism of tryptophan and is able to degrade the tryptophan-byproducts kynurenine and hydroxy-kynurenine (OH-Kyn) into anthranilic acid and hydroxy-anthranilic acid (OH-AA), respectively. Enzyme-mediated modulation of tryptophan metabolism may be of therapeutic relevance7.

<meta charset="utf8"></head><body>Autor em destaque: Avançando na engenharia celular e de proteínas para controlar funções biológicas e desenvolver novas terapias

Determinando a estabilidade sérica da enzima adenosina desaminase 1 humana

So our lab works at the interface of immunology, engineering, and metabolism to improve human health. We use our expertise in protein and cellular engineering to make new therapies and control biological functions. If researchers desire to increase the circulating half-life of protein biologics, this protocol could help inform users whether serum stability is a rate limiting step.Understanding this key point would help guide future protein engineering efforts to the area of need. One question we'll explore is whether proteins with enhanced serum stability result in greater therapeutic efficacy in the context of treating cancer than their non engineered counterparts. We'll further explore whether therapeutic efficacy can be achieved using less doses due to this enhanced stability.

The concept of enzyme stability is typically used to refer to an enzyme's thermostability - its ability to retain structure and activity as temperature ...

determining-the-serum-stability-of-human-adenosine-deaminase-1-enzyme

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Determining the Serum Stability of Human Adenosine Deaminase 1 Enzyme

JoVE Journal

Bioengineering

348 visualizações.  Georgia Institute of Technology. Portanto, nosso laboratório trabalha na interface de imunologia, engenharia e metabolismo para melhorar a saúde humana. Usamos nossa experiência em engenharia de proteínas e células para fazer novas terapias e controlar funções biológicas. Se os pesquisadores desejam aumentar a meia-vida circulante dos produtos biológicos de proteínas, este protocolo pode ajudar a informar aos usuários se a estabilidade sérica é uma etapa limitante da taxa.