This study was supported by the open fund of state key laboratory of Pharmaceutical Biotechnology, Nan-jing University, China (Grant no. KF-GN-201905), the National Natural Science Foundation of China (grants 81971501). William J. Liu is supported by the Excellent Young Scientist Program of the NSFC (81822040) and Beijing New-star Plan of Science and Technology (Z181100006218080).

1. Preparation of expression constructs
<ol>
	<li>Retrieve the sequences of MHC class I genes (including predicted genes) from bats from the NCBI database.</li>
	<li>Retrieve higher mammal MHC I heavy chain sequences from the Immuno Polymorphism Database (IPD) (www.ebi.ac.uk/ipd/mhc) and the UniProt database (www.uniprot.org).</li>
	<li>To obtain soluble MHC complexes, mutagenize the sequences to remove the cytosolic and transmembrane regions.</li>
	<li>Clone the genes encoding the ectodomains of the bat Ptal-N*01:01 (...

<ol>
	<li>Vyas, J. M., Van der Veen, A. G., Ploegh, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+known+unknowns+of+antigen+processing+and+presentation.">The known unknowns of antigen processing and presentation.</a> Nature Reviews Immunology. 8 (8), 607-618 (2008).</li><li>Bjorkman, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+human+class+I+histocompatibility+antigen,+HLA-A2.">Structure of the human class I histocompatibility antigen, HLA-A2.</a> Nature. 329 (6139), 506-512 (1987).</li><li>Seong, R. H., Clayberger, C. A., Krensky, A. M., Parnes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+Daudi+cell+HLA+expression+by+transfection+of+the+mouse+beta+2-microglobulin+gene.">Rescue of Daudi cell HLA expression by transfection of the mouse beta 2-microglobulin gene.</a> Journal of Experimental Medicine. 167 (2), 288-299 (1988).</li><li>Zijlstra, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta+2-microglobulin+deficient+mice+lack+CD4-8++cytolytic+T+cells.">Beta 2-microglobulin deficient mice lack CD4-8+ cytolytic T cells.</a> Nature. 344 (6268), 742-746 (1990).</li><li>Gao, G. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+complex+between+human+CD8alpha(alpha)+and+HLA-A2.">Crystal structure of the complex between human CD8alpha(alpha) and HLA-A2.</a> Nature. 387 (6633), 630-634 (1997).</li><li>Bjorkman, P. J., Parham, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+function,+and+diversity+of+class+I+major+histocompatibility+complex+molecules.">Structure, function, and diversity of class I major histocompatibility complex molecules.</a> Annual Review of Biochemistry. 59, 253-288 (1990).</li><li>Achour, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+the+differential+stability+and+receptor+specificity+of+H-2Db+in+complex+with+murine+versus+human+beta+2-microglobulin.">Structural basis of the differential stability and receptor specificity of H-2Db in complex with murine versus human beta 2-microglobulin.</a> Journal of Molecular Biology. 356 (2), 382-396 (2006).</li><li>Kubota, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+serum+beta+2-microglobulin+with+H-2+class+I+heavy+chains+on+the+surface+of+mouse+cells+in+culture.">Association of serum beta 2-microglobulin with H-2 class I heavy chains on the surface of mouse cells in culture.</a> Journal of Immunology. 133 (6), 3203-3210 (1984).</li><li>Bernabeu, C., van de Rijn, M., Lerch, P. G., Terhorst, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta+2-microglobulin+from+serum+associates+with+MHC+class+I+antigens+on+the+surface+of+cultured+cells.">Beta 2-microglobulin from serum associates with MHC class I antigens on the surface of cultured cells.</a> Nature. 308 (5960), 642-645 (1984).</li><li>Lu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+presentation+by+bat+MHC+class+I+provides+new+insight+into+the+antiviral+immunity+of+bats.">Peptide presentation by bat MHC class I provides new insight into the antiviral immunity of bats.</a> PLoS Biology. 17 (9), 3000436 (2019).</li><li>Wu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+diverse+peptide+accommodation+by+the+rhesus+macaque+MHC+class+I+molecule+Mamu-B*17:+insights+into+immune+protection+from+simian+immunodeficiency+virus.">Structural basis of diverse peptide accommodation by the rhesus macaque MHC class I molecule Mamu-B*17: insights into immune protection from simian immunodeficiency virus.</a> Journal of Immunology. 187 (12), 6382-6392 (2011).</li><li>Chu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+glimpse+of+the+peptide+presentation+by+rhesus+macaque+MHC+class+I:+crystal+structures+of+Mamu-A*01+complexed+with+two+immunogenic+SIV+epitopes+and+insights+into+CTL+escape.">First glimpse of the peptide presentation by rhesus macaque MHC class I: crystal structures of Mamu-A*01 complexed with two immunogenic SIV epitopes and insights into CTL escape.</a> Journal of Immunology. 178 (2), 944-952 (2007).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+peptide+presentation+of+rhesus+macaque+major+histocompatibility+complex+class+I+Mamu-A*02+revealed+by+two+peptide+complex+structures+and+insights+into+immune+escape+of+simian+immunodeficiency+virus.">Diverse peptide presentation of rhesus macaque major histocompatibility complex class I Mamu-A*02 revealed by two peptide complex structures and insights into immune escape of simian immunodeficiency virus.</a> Journal of Virology. 85 (14), 7372-7383 (2011).</li><li>Liu, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+T+cell+responses+featured+by+concordant+recognition+of+Middle+East+respiratory+syndrome+coronavirus-derived+CD8++T+cell+epitopes+and+host+MHC.">Protective T cell responses featured by concordant recognition of Middle East respiratory syndrome coronavirus-derived CD8+ T cell epitopes and host MHC.</a> Journal of Immunology. 198 (2), 873-882 (2017).</li><li>Mitaksov, V., Fremont, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+definition+of+the+H-2Kd+peptide-binding+motif.">Structural definition of the H-2Kd peptide-binding motif.</a> Journal of Biological Chemistry. 281 (15), 10618-10625 (2006).</li><li>Xiao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversified+anchoring+features+the+peptide+presentation+of+DLA-88*50801:+first+structural+insight+into+domestic+dog+MHC+class+I.">Diversified anchoring features the peptide presentation of DLA-88*50801: first structural insight into domestic dog MHC class I.</a> Journal of Immunology. 197 (6), 2306-2315 (2016).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+conformations+of+a+rinderpest+virus+epitope+presented+by+bovine+major+histocompatibility+complex+class+I+N*01801:+a+host+strategy+to+present+featured+peptides.">Two distinct conformations of a rinderpest virus epitope presented by bovine major histocompatibility complex class I N*01801: a host strategy to present featured peptides.</a> Journal of Virology. 85 (12), 6038-6048 (2011).</li><li>Yao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+illumination+of+equine+MHC+class+I+molecules+highlights+unconventional+epitope+presentation+manner+that+is+evolved+in+equine+leukocyte+antigen+alleles.">Structural illumination of equine MHC class I molecules highlights unconventional epitope presentation manner that is evolved in equine leukocyte antigen alleles.</a> Journal of Immunology. 196 (4), 1943-1954 (2016).</li><li>Wynne, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+antigen+processing+machinery+and+endogenous+peptide+presentation+of+a+bat+MHC+class+I+molecule.">Characterization of the antigen processing machinery and endogenous peptide presentation of a bat MHC class I molecule.</a> Journal of Immunology. 196 (11), 4468-4476 (2016).</li><li>Zhang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+cross-allele+presentation+by+HLA-A*0301+and+HLA-A*1101+revealed+by+two+HIV-derived+peptide+complexes.">Structural basis of cross-allele presentation by HLA-A*0301 and HLA-A*1101 revealed by two HIV-derived peptide complexes.</a> Molecular Immunology. 49 (1-2), 395-401 (2011).</li><li>Hoof, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NetMHCpan,+a+method+for+MHC+class+I+binding+prediction+beyond+humans.">NetMHCpan, a method for MHC class I binding prediction beyond humans.</a> Immunogenetics. 61 (1), 1-13 (2009).</li><li>Raveh, B., London, N., Zimmerman, L., Schueler-Furman, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rosetta++FlexPepDock+ab-initio:+simultaneous+folding,+docking+and+refinement+of+peptides+onto+their+receptors.">Rosetta FlexPepDock ab-initio: simultaneous folding, docking and refinement of peptides onto their receptors.</a> PLoS One. 6 (4), 18934 (2011).</li><li>Otwinowski, Z., Minor, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+X-ray+diffraction+data+collected+in+oscillation+mode.">Processing of X-ray diffraction data collected in oscillation mode.</a> Methods in Enzymology. 276, 307-326 (1997).</li><li>Brunger, A. 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The authors have nothing to declare.

The construction of a hybrid protein complex through heterologous substitution from different taxa is a common strategy for functional and structural investigations when the homologous complex is not available, such as in the MHC I and its ligands. However, there is limited summarization regarding the methodology and the technology. Herein, the structure of bat MHC I, Ptal-N*01:01, stabilized by b&#946;2m or h&#946;2m was analyzed. The key amino acids of &#946;2m binding to Ptal-N*01:01 w...

The major histocompatibility complex (MHC) exists in all vertebrates and is a set of genes that determines the cell-mediated immunity to infectious pathogens. MHC class I presents endogenous peptides, such as viral components produced upon virus infection, to T cell receptors (TCR) on the surface of CD8+ T cells to mediate cellular immunity and participate in immune regulation1. A structural study of MHC I binding to peptides provides information regarding peptide binding motifs and presentation features by MHC I molecules, which plays vital roles in evaluation of CD8+ T cell immune responses and vaccine development.
Since the first crystallization and structural determination of MHC I molecular by Bjorkman et al.2, the crystal structure analysis of MHC I molecules has greatly promoted the understanding of how peptides bind to MHC I molecules, and helps to understand the interaction of light chains with heavy chains and peptides. A series of follow-up studies indicated that although the genes encoding the light chain is not associated with the MHC, the light chain is a key protein for the assembly of MHC I molecules3,4. It interacts with the three domains of MHC class I molecules on multiple surfaces. When the light chain is absent, MHC class I molecules cannot be correctly expressed on the surface of antigen-presenting cells and cannot interact with TCR to exert their immunological functions.
MHC I is comprised of a heavy chain (H chain) and light chain (i.e., &#946;2-microglobulin (&#946;2m)), and is assembled through binding to a suitable peptide5. The extracellular segment of the H chain consists of &#945;1, &#945;2 and &#945;3 domains6. The &#945;1 and &#945;2 domains form the peptide binding groove (PBG). The &#946;2m chain acts as a structural subunit of the assembly complex in MHC I, stabilizing the conformation of the complex, and is a molecular chaperone for MHC I H chain folding7,8,9. A series of studies have shown that MHC I H chains from various mammals such as bat (Chiroptera) (Ptal-N*01:01)10, rhesus macaque (Primates) (Mamu-B*17)11 (Mamu-A*01)12 (Mamu-A*02)13, mouse (Rodentia) (H-2Kd)14,15, dog (Carnivora) (DLA-88*50801)16, cattle (Artiodactyla) (BoLA-A11)17 and equine (Perissodactyla) (Eqca-N*00602 and Eqca-N*00601)18 can combine with heterologous &#946;2m (Table 1). These hybrid molecules are often used in structural and functional studies. However, the methodology for the functional and structural study of the hybrid MHC I with heterologous &#946;2m is not yet summarized. Meanwhile, the structural basis for the interchanged &#946;2m between different taxa remain unclear.
Herein, the procedure for MHC I expression, refolding, crystallization, crystal data collection and structure determination are summarized. In addition, potential substitutions of &#946;2m from different species are analyzed through comparing the structural conformation of MHC I stabilized by homologous and heterologous &#946;2m. These methods will be helpful for further MHC I structural study and CD8+ T cell immune response evaluation in cancer and infectious disease.

<table>
	<tbody>
		<tr>
			<td>10 kDa MMCO membrane</td>
			<td>Merck millipore</td>
			<td>PLGC07610</td>
			<td></td>
		</tr>
		<tr>
			<td>30% Acrylamide</td>
			<td>LABLEAD</td>
			<td>A3291-500ml*5</td>
			<td></td>
		</tr>
		<tr>
			<td>5&#215;Protein SDS Loading</td>
			<td>Novoprotein</td>
			<td>PM099-01A</td>
			<td></td>
		</tr>
		<tr>
			<td>AMICON ULTRA-15 15ML-10 KDa cutoff</td>
			<td>Merck millipore</td>
			<td>UFC901096</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Inalco</td>
			<td>1758-9314</td>
			<td></td>
		</tr>
		<tr>
			<td>APS</td>
			<td>Sigma</td>
			<td>A3678-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21(DE3) strain</td>
			<td>TIANGEN</td>
			<td>CB105-02</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>MP</td>
			<td>219605580</td>
			<td>Wear suitable gloves and eye/face protection. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Solarbio</td>
			<td>D1070</td>
			<td>Gloves and goggles should be worn and operated in a ventilated kitchen. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.</td>
		</tr>
		<tr>
			<td>EDTA-2Na</td>
			<td>KeyGEN BioTECH</td>
			<td>KGT515500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerin</td>
			<td>HUSHI</td>
			<td>10010618</td>
			<td></td>
		</tr>
		<tr>
			<td>GSH</td>
			<td>Amresco</td>
			<td>0399-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>GSSG</td>
			<td>Amresco</td>
			<td>0524-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanidine hydrochloride</td>
			<td>Amresco</td>
			<td>E424-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>h&#946;2m</td>
			<td>our lab</td>
			<td></td>
			<td>Zhang, S. et al. Structural basis of cross-allele presentation by HLA-A*0301 and HLA-A*1101 revealed by two HIV-derived peptide complexes. Mol Immunol. 49 (1-2), 395-401, (2011).</td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Inalco</td>
			<td>1758-1400</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Arginine Hydrochloride</td>
			<td>Amresco</td>
			<td>0877-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Solarbio</td>
			<td>S8210</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Marker</td>
			<td>Fermentas</td>
			<td>26614</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Boao Rui Jing</td>
			<td>A112130</td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex Increase 200 10/300 GL</td>
			<td>GE Healthcare</td>
			<td>28990944</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Thermo</td>
			<td>17919</td>
			<td>Gloves and goggles should be worn and operated in a ventilated kitchen.</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Amresco</td>
			<td>0497-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Bioruler</td>
			<td>RH30056-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Oxoid</td>
			<td>LP0042</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Oxoid</td>
			<td>LP0021</td>
			<td></td>
		</tr>
	</tbody>
</table>

stability and structure of bat major histocompatibility complex class i with heterologous &beta;2-microglobulin

The major histocompatibility complex (MHC) plays a pivotal role in antigen peptide presentation and T cell immune responses against infectious disease and tumor development. The hybrid MHC I complexed with heterologous &#946;2-microglobulin (&#946;2m) substitution from different species can be stabilized in vitro. This is a feasible means to study MHC I of mammals, when the homologous &#946;2m is not available. Meanwhile, it is indicated that mammalian &#946;2m substitution does not significantly affect peptide presentation. However, there is limited summarization regarding the methodology and the technology for the hybrid MHC I complexed with heterologous &#946;2-microglobulin (&#946;2m). Herein, methods to evaluate the feasibility of heterologous &#946;2m substitution in MHC I study are presented. These methods include preparation of expression constructs; purification of inclusion bodies and refolding of the MHC complex; determination of protein thermostability; crystal screening and structure determination. This study provides a recommendation for understanding function and structure of MHC I, and is also significant for T cell response evaluation during infectious disease and tumor immunotherapy.

Previous work reported that the HeV-derived HeV1 (DFANTFLP) peptide was presented by Ptal-N*01:0110,19. Herein, the binding capacity of this peptide to Ptal-N*01:01 with homologous bat &#946;2m (b&#946;2<font face="Helvetica Neue...

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Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous &amp;#946;2-Microglobulin

The protocol describes experimental methods to obtain stable major histocompatibility complex (MHC) class I through potential &#946;2-microglobulin (&#946;2m) substitutions from different species. The structural comparison of MHC I stabilized by homologous and heterologous &#946;2m were investigated.

Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous &beta;2-Microglobulin

The major histocompatibility complex (MHC) plays a pivotal role in antigen peptide presentation and T cell immune responses against infectious disease and ...

In this study, we used Bat MHC Class I as a model to summarize the methodology, and to evaluate the feasibility of a hybrid MHC class I complexed with heterologous beta-2 microglobulin. Previous studies have shown that the mammalian B2M substitution does not significantly affect the peptide presentation. In fact, the hybrid MHC Class I can activate a similar structure and function to the original molecule.These techniques can be used to facilitate the functional and structured study of MHC I for tissue response evaluation during infectious disease and tumor immunotherapy. For E.coli culture transformation add 10 nanograms of plasmid containing bat or human MHC Class I beta two microglobulin hydrogen chain to 100 microliters of equalized suspension and bathe the suspension in ice for 30 minutes. At the end of the incubation, heat shock the bacteria for 90 seconds at 42 degrees Celsius, and return the culture to the ice bath for two minutes.Next add 800 microliters of lysogeny broth to the culture, and shake the suspension on a rocking platform for 20 minutes at 37 degrees Celsius, and 200 rotations per minute. At the end of the incubation, spread 100 microliters of the bacteria onto an appropriate antibiotic resistant culture blade. The next morning, pick a single recently transformed bacterial clone and inoculate the clone with three milliliters of LB with antibiotic medium.Place the culture on the rocking platform at 37 degrees Celsius for 12 to 16 hours. The next morning, transfer 500 microliters of the activated bacterial stock into 50 milliliters of LB with antibiotic medium, and return the bacteria to the rocking incubator overnight. Split the remaining activated bacterial stock into 500 microliter aliquots and add each aliquot to 500 microliter volumes of 40%glycerol, for minus 80 degrees Celsius storage.To generate large amounts of recombinant protein, the next morning transfer the bacterial suspension into two liters of LB with antibiotic medium at a one to 100 ratio, and return the culture to the rocking incubator until an absorbance of 0.6 at 600 nanometers is achieved. Then, add one millimolar IPTG to the culture to induce expression of the trans gene. After four to six hours of induction, transfer the bacterial culture to bottles for centrifugation, and re-suspend the bacterial cell pellets in 60 milliliters of PBS per bottle.Then liberate the expressed recombinant protein by ultrasonic cell disruptor 99 times, for six seconds and an interval of 12 seconds at 300 Watts per sonication. For inclusion body purification collect the sonicated bacteria by centrifugation and re-suspend the pellets in an appropriate volume of washing buffer for two additional centrifugations. After the second wash, re-suspend the inclusion bodies and re-suspension buffer, and set aside a 20 microliter aliquot of the sample for SDS page to test the inclusion body purity.Centrifuge the remaining sample, and weigh the inclusion body containing pellet. Add dissolution buffer to the pellets to a final concentration of 30 milligrams of inclusion body per milliliter of buffer. And use a magnetic stirrer to slowly stir the solution at four degrees Celsius, until the inclusion bodies are dissolved in the dissolution buffer.After discarding the precipitates, store the inclusion bodies at minus 20 or minus 80 degrees Celsius. For MHC complex refolding, add a five millimolar reduced glutathione and 0.5 millimolar oxidized glutathione at 250 to 300 milliliters, a four degrees Celsius refolding buffer. And slowly stir the solution on a magnetic stirrer at four degrees Celsius for 10 to 20 minutes.For MHC hydrogen chain and beta two microglobulin dilution, load the inclusion body in a one milliliter syringe and inject the entire one milliliter volume of inclusion body solution into one liter of refolding buffer near the stir bar, to obtain a fast and efficient dilution. Next, dissolve five milligrams per milliliter of peptide in dimethyl sulphoxide, and quickly inject 200 microliters of peptide into the refolding solution as just demonstrated. After 10 to 20 minutes of slow stirring, inject three milliliters of H chain inclusion bodies into a new one liter volume of refolding buffer and allow the refolding to proceed at four degrees Celsius for eight to 10 hours.To determine the refolded protein concentration add exchange buffer to a pressurized chamber with a 10 kilodalton multi copper oxidase membrane, and concentrate the buffer to 30 to 50 milliliter volume. Transfer the refolding solution to a centrifuge tube, and remove the precipitates by centrifugation. Carefully transfer the supernate into the chamber and further concentrate the buffer to a final volume of approximately one milliliter.Remove any final contaminants by centrifugation and transfer the supernate into a sterile tube. Use a ten three hundred GL size exclusion column to purify the proteins, collecting the samples at the peak, and analyzing them using SDS page. Collect the MHC complex peak and concentrate the protein to a final concentration of 15 milligrams per milliliter.Then dilute the complex to 7.5 milligrams per milliliter concentration. To perform crystallization of the complex MHC and peptide using the sitting drop vapor diffusion technique, add 0.8 microliters of the sample to a commercial crystal board and seal the board. Equilibrate the sample solution against 100 microliters of reservoir solution at four or 18 degrees Celsius, and use a microscope to regularly observe the crystal growth over the next six months.For cryogenic protection, at the end of the experiment, transfer the crystals to a storage solution containing 20%glycerol before rapidly cooling the samples in a 100 degree Kelvin gaseous nitrogen stream. Then collect x-ray diffraction data according to standard protocols. To determine the structure of the crystal complexes, open the high resolution structural x-ray diffraction data in the Denzo program within the HK L 2000 software package, and select an initial model in the protein data bank.To determine the structure of the protein, open the data in the Phaser MR Program in CCP4, and use the refined x-ray model for the initial joint refinement. Then use the Phoenix refine alone and joint modes for the x-ray alone refinement, and the joint neutron for the x-ray refinement. After each round of refinement, manually check the model against the Fo-Fc, and 2Fo-Fc positive nuclear density maps in Coot.In these representative experiments, the binding capacities of the Hendra Virus derived Hendra Virus One peptide to bat MHC Class I hydrogen chains with homologous bat beta two microglobulin and heterologous human beta two microglobulin were evaluated. In both analysis, crystals with a high resolution were formed through renaturation with the beta two microglobulin light chain. In the absence of beta two microglobulin, these complexes do not form.In the MHC Class I hydrogen chain Hendra Virus one, human beta two microglobulin structure, conserved residues H31, D53, W60, and Y63 of human beta two microglobulin, which correspond to bat beta two microglobulin, make contact with the bottom of the peptide binding groove and conserved Q8, Y10, R12, and 24 residues, which correspond to the beta two microglobulins bound to the alpha three domain. In the overall bat and human structures, the average root mean square derivation of residues one to 184 of the hydrogen chains is 0.248 under all of the carbon alpha atom's superpositions, indicating that there are no differences between these two complexes. The structure of the peptide alignment shows that the confirmations of Hendra Virus one peptides in these two complexes are quite similar.Sequence alignment also shows that the amino acids of beta two microglobulin from different species are highly conserved. It is important to offer the adversary complexes with a high reporting efficiency. Therefore, it is crucial to select suitable beta two micro globulin and to use highly purified inclusion bodies.This protocol can be used to obtain stable MTC I complex through potential beta two microglobulin substitutions in other species, and to exist they are MTC I structure and function. The data obtained from these studies can be used to assess T-cell responses during infectious disease and tumor immunotherapies.

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JoVE Journal

Immunology and Infection

6.2K Görüntüleme.  Wenzhou Medical University. Bu çalışmada, metodolojiyi özetlemek ve heterolog beta-2 mikroglobulin ile karmaşık hale gelen hibrit bir MHC sınıfının fizibilitesini değerlendirmek için model olarak Bat MHC Class I'i kullandık. Önceki çalışmalar, memeli B2M ikamesinin peptit sunumını önemli ölçüde etkilemediğini göstermiştir. Aslında, hibrid MHC Sınıfı I, orijinal moleküle benzer bir yapıyı ve işlevi etkinleştirebilir.Bu teknikler, bulaşıcı hastalık ve tümör immünoterapisi s...