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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-. </p></div></div></div>
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<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK594022_"><span class="title" itemprop="name">Recombinant protein preparation and functional analysis of mannan-binding protein</span></h1><div class="contrib half_rhythm"><span itemprop="author">Motohiro Nonaka</span>, Dr.<div class="affiliation small">Kyoto University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.u-otoyk@r4.orihotom.akanon" class="oemail">pj.ca.u-otoyk@r4.orihotom.akanon</a></div></div><div class="small">Corresponding author.</div></div><div class="contrib half_rhythm"><span itemprop="author">Toshisuke Kawasaki</span>, Dr.<div class="affiliation small">Ritsumeikan University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.iemustir.cf@kasawakt" class="oemail">pj.ca.iemustir.cf@kasawakt</a></div></div></div><p class="small">Created: <span itemprop="datePublished">October 1, 2021</span>; Last Revision: <span itemprop="dateModified">March 21, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g98-recmanbinpro.Introduction"><h2 id="_g98-recmanbinpro_Introduction_">Introduction:</h2><p>To perform reproducible functional analysis of glycoproteins in immunology and oncology, homogeneous and highly pure proteins are required. Various heterologous expression systems, such as viral and bacterial vectors, have been commonly used to produce recombinant proteins.</p><p>Human mannan-binding protein (MBP) is expressed in the liver and is classified according to its subsequent localization: MBP remaining in the liver cells (L-MBP) and MBP being secreted into the serum (S-MBP). L-MBP, now also known as intracellular MBP (I-MBP), functions in the quality control of newly-synthesized glycoproteins in hepatocytes (<a class="bk_pop" href="#g98-recmanbinpro.REF.1">1</a>). S-MBP functions in the innate immunity against colorectal cancers (<a class="bk_pop" href="#g98-recmanbinpro.REF.2">2</a>–<a class="bk_pop" href="#g98-recmanbinpro.REF.5">5</a>) and exogenous ligands. A sufficient number of multimerization is necessary for S-MBP to function successfully (<a class="bk_pop" href="#g98-recmanbinpro.REF.6">6</a>). Therefore, correct selection of the expression system (e.g., cell lines) is a critical step in the preparation of recombinant protein.</p></div><div id="g98-recmanbinpro.Protocol"><h2 id="_g98-recmanbinpro_Protocol_">Protocol</h2><p>In this protocol, a vaccinia virus expression system was used to express recombinant human MBP in human hepatocytes. Complement activation assay demonstrated that this recombinant protein is structurally and functionally similar to native human MBP purified from human plasma samples (<a class="figpopup" href="/books/NBK594022/figure/g98-recmanbinpro.F1/?report=objectonly" target="object" rid-figpopup="figg98recmanbinproF1" rid-ob="figobg98recmanbinproF1">Figure 1</a>).</p><div id="g98-recmanbinpro.Materials"><h3>Materials</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">The human MBP cDNA</p></dd><dt>2.</dt><dd><p class="no_top_margin">A vaccinia virus transfer vector, pBSF-2-16</p></dd><dt>3.</dt><dd><p class="no_top_margin">Wild-type vaccinia virus strain Western Reserve (WR) and its isatin-β-thiosemicarbazone-(IBT)-dependent derivative</p></dd><dt>4.</dt><dd><p class="no_top_margin">RK13 (a rabbit kidney cell line, ATCC CCL 37), COS-7 (an African green monkey kidney cell line, ATCC CRL 1651), and HLF (a human hepatoma cell line, JCRB 0405)</p></dd><dt>5.</dt><dd><p class="no_top_margin">All materials for cell culture, virus proliferation, vector construction and transfection, virus purification, and recombinant protein purification</p></dd><dt>6.</dt><dd><p class="no_top_margin">An affinity column of mannan-Sepharose 4B and gel filtration chromatography with Sephacryl S-300</p></dd><dt>7.</dt><dd><p class="no_top_margin">Loading buffer: 20 mM of imidazole, 1.25 M NaCl, and 20 mM of CaCl<sub>2</sub>, pH 7.8</p></dd><dt>8.</dt><dd><p class="no_top_margin">Elution buffer: 20 mM of imidazole, 1.25 M NaCl, and 4 mM of EDTA, pH 7.8</p></dd><dt>9.</dt><dd><p class="no_top_margin">All chemicals for gel electrophoresis</p></dd><dt>10.</dt><dd><p class="no_top_margin">All reagents for MBP complement activation assay</p></dd><dt>11.</dt><dd><p class="no_top_margin">Gelatin-veronal buffer (GVB): 5 mM of veronal buffer, pH 7.4, containing 0.145 M NaCl, 0.1% gelatin, 2 mM of CaCl<sub>2</sub>, and 0.5 mM of MgCl<sub>2</sub></p></dd></dl></div><div id="g98-recmanbinpro.Instruments"><h3>Instruments</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Additional reagents and equipment for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), immunodetection, affinity column purification, gel filtration chromatography, and MBP complement activation assay</p></dd></dl></div><div id="g98-recmanbinpro.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Construction of a vaccinia virus transfer vector and recombinant vaccinia virus (RVV) (<b>Note 1</b>)</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">The human MBP cDNA is excised from the vector by digestion with SmaI and SacI and is subcloned into a vaccinia virus transfer vector, pBSF2-16.</p></dd><dt>b.</dt><dd><p class="no_top_margin">In the resultant transfer vector (pBSF2-16/MBP), the human MBP cDNA is located immediately downstream of the A-type inclusion body of a cowpox virus (ATI) hybrid promoter, and the human MBP cDNA flanked by the ATI hybrid promoter is interposed in the hemagglutinin (HA) gene, a selection marker for obtaining RVVs.</p></dd><dt>c.</dt><dd><p class="no_top_margin">pBSF2-16/MBP (5 µg) and intact genomic DNA (10 µg) extracted from wild-type WR vaccinia virus are diluted in 1.5 mL of OptiMEM, and 30 μg of a cationic liposomal transfection reagent is diluted in 1.5 mL of OptiMEM. These two solutions are then mixed gently and incubated at room temperature for 45 min to yield the DNA-liposome complex.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Cultured COS-7 cells (2.5 × 10<sup>5</sup> cells in a 10-cm dish) are infected with 2.5 × 10<sup>4</sup> plaque-forming units of IBT-dependent vaccinia virus for 1 h. The cells are exposed to the transfection solution for 8 h at 37°C under 5% CO<sub>2</sub>.</p></dd><dt>e.</dt><dd><p class="no_top_margin">After 20 h of incubation, the cells are harvested, and then the virus progeny is released by sonication. Monolayer culture of RK13 cells is infected with the progeny virus.</p></dd><dt>f.</dt><dd><p class="no_top_margin">The virus progeny obtained from HA-negative plaques inoculated onto monolayer culture of RK13 cells and further screened for the expression of human MBP by immunohistochemical staining with a monoclonal antibody specific for human MBP.</p></dd><dt>g.</dt><dd><p class="no_top_margin">The virus progeny obtained from MBP-positive plaques is selected once more to purify the RVV.</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">Expression and purification of recombinant human MBP</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">HLF cells at a density of 1 × 10<sup>7</sup> cells per 75 cm<sup>2</sup> are infected with the RVV containing the human MBP cDNA at a multiplicity of infection of 5, and then the infected cells are incubated for 48 h at 37°C under 5% CO<sub>2</sub> (<b>Note 2</b>).</p></dd><dt>b.</dt><dd><p class="no_top_margin">The culture supernatant of HLF cells is harvested and centrifuged to remove cell debris and viral particles.</p></dd><dt>c.</dt><dd><p class="no_top_margin">The resulting supernatant is mixed with a half volume 3 × loading buffer, filtered through a 0.45-μm filter, and then applied to a mannan-Sepharose 4B column equilibrated with loading buffer.</p></dd><dt>d.</dt><dd><p class="no_top_margin">After washing the column with loading buffer, the bound recombinant proteins are eluted with elution buffer.</p></dd><dt>e.</dt><dd><p class="no_top_margin">The eluted fractions are pooled and concentrated. The purity of the recombinant MBP can be checked by SDS-PAGE under reducing conditions, and the degree of oligomerization is assessed by SDS-PAGE on a 3%–10% gradient gel under nonreducing conditions. A typical yield of recombinant human MBP is 5 mg/L of culture medium.</p></dd></dl></dd><dt>3.</dt><dd><p class="no_top_margin">Assay for complement activation activity of MBP by passive hemolysis</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">The sheep erythrocyte suspension is centrifuged at 350 ×g for 5 min at 4°C and suspend with saline several times.</p></dd><dt>b.</dt><dd><p class="no_top_margin">A portion of the suspension is taken, and the erythrocyte is lysed with water.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Determine the cell density of an erythrocyte suspension in (a) by measuring the absorbance at 541 nm.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Mix 1 mL of 200 μg/mL mannan solution with 1 mL of a 0.5 mg/mL CrCl3 solution.</p></dd><dt>e.</dt><dd><p class="no_top_margin">The mixture is added to 2 mL of a 1 × 10<sup>9</sup> cells/mL erythrocyte suspension, followed by incubation for 5 min at room temperature with occasional mixing.</p></dd><dt>f.</dt><dd><p class="no_top_margin">The reaction is stopped by adding 3 mL of ice-cold GVB buffer.</p></dd><dt>g.</dt><dd><p class="no_top_margin">The resulting erythrocytes coated with mannan (mannan-erythrocytes, ME) are washed with the GVB buffer thrice and then resuspended with GVB buffer at a density of 1 × 10<sup>9</sup> cells/mL.</p></dd><dt>h.</dt><dd><p class="no_top_margin">Mix 0.1 mL of ME suspension and 0.4 mL of recombinant human MBP diluted with GVB buffer and incubated with gentle shaking to sensitize ME with MBP.</p></dd><dt>i.</dt><dd><p class="no_top_margin">The ME suspension is then washed with ice-cold GVB buffer and resuspended at a density of 1 × 10<sup>9</sup> cells/mL. Two volumes of CH50 of MBP-depleted guinea pig complement (<b>Note 3</b>), 0.1 mL of the ME suspension sensitized with MBP, and GVB buffer are mixed in a total volume of 1.5 mL on ice and are then incubated for 1 h at 37°C.</p></dd><dt>j.</dt><dd><p class="no_top_margin">After the reaction mixture has been centrifuged, the absorbance at 541 nm of the supernatant is measured. Maximal lysis is obtained by incubation of 0.1 mL of the ME suspension with 0.14 mL of water. The degree of specific lysis is expressed as a percentage of the maximal lysis (<b>Note 4</b>).</p></dd></dl></dd></dl></div><div id="g98-recmanbinpro.Notes"><h3>Notes</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Vaccinia viruses are DNA viruses that infect almost all mammalian cells. The use of vaccinia virus as a vector to introduce cDNAs into mammalian cells has several useful advantages, including a relatively high level of protein synthesis, proper folding, disulfide bond formation, glycosylation, and other post-translational modifications. Thus, a vaccinia virus expression system is useful for the recombinant preparation of highly assembled macromolecules, such as MBP.</p></dd><dt>2.</dt><dd><p class="no_top_margin">Recombinant human MBPs have been produced in myeloma cells, COS cells, and CHO cells. However, these recombinant MBPs contain oligomers with fewer polypeptide chains than those found in native MBP and exhibit less ability to active complement compared to native MBP.</p></dd><dt>3.</dt><dd><p class="no_top_margin">A CH50 unit is defined as the amount of complement solution that gives a 50% hemolysis of 1 × 10<sup>9</sup> cells/mL erythrocytes.</p></dd><dt>4.</dt><dd><p class="no_top_margin">The complement activation activity of MBP is assayed by passive hemolysis using mannan-coated sheep erythrocytes in the presence of complement. The binding of MBP to the surfaces of mannan-coated erythrocytes triggers the complement cascade, resulting in the formation of membrane attack complexes and cell lysis. The degree of lysis is determined by hemoglobin colorimetry. The complement activation activity of recombinant human MBP increases with the degree of oligomerization.</p></dd></dl></div></div><div id="g98-recmanbinpro.References"><h2 id="_g98-recmanbinpro_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g98-recmanbinpro.REF.1">Nonaka M, Ma BY, Ohtani M, Yamamoto A, Murata M, Totani K, et al. Subcellular localization and physiological significance of intracellular mannan-binding protein. <span><span class="ref-journal">J Biol Chem. </span>2007 Jun 15;<span class="ref-vol">282</span>(24):17908–20.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17442667" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17442667</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.M700992200" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g98-recmanbinpro.REF.2">Ma Y, Uemura K, Oka S, Kozutsumi Y, Kawasaki N, Kawasaki T. Antitumor activity of mannan-binding protein in vivo as revealed by a virus expression system: mannan-binding proteindependent cell-mediated cytotoxicity. <span><span class="ref-journal">Proc Natl Acad Sci U S A. </span>1999 Jan 19;<span class="ref-vol">96</span>(2):371–5.</span> [<a href="/pmc/articles/PMC15143/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC15143</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/9892640" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9892640</span></a>] [<a href="http://dx.crossref.org/10.1073/pnas.96.2.371" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g98-recmanbinpro.REF.3">Terada M, Khoo K-H, Inoue R, Chen C-I, Yamada K, Sakaguchi H, et al. Characterization of oligosaccharide ligands expressed on SW1116 cells recognized by mannan-binding protein. A highly fucosylated polylactosamine type N-glycan. <span><span class="ref-journal">J Biol Chem. </span>2005 Mar 25;<span class="ref-vol">280</span>(12):10897–913.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/15634673" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15634673</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.M413092200" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="g98-recmanbinpro.REF.4">Kawasaki N, Lin C-W, Inoue R, Khoo K-H, Kawasaki N, Ma BY, et al. Highly fucosylated N-glycan ligands for mannan-binding protein expressed specifically on CD26 (DPPVI) isolated from a human colorectal carcinoma cell line, SW1116. <span><span class="ref-journal">Glycobiology. </span>2009 Apr;<span class="ref-vol">19</span>(4):437–50.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19129245" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19129245</span></a>] [<a href="http://dx.crossref.org/10.1093/glycob/cwn158" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="g98-recmanbinpro.REF.5">Nonaka M, Imaeda H, Matsumoto S, Yong Ma B, Kawasaki N, Mekata E, et al. Mannan-binding protein, a C-type serum lectin, recognizes primary colorectal carcinomas through tumor-associated Lewis glycans. <span><span class="ref-journal">J Immunol. </span>2014 Feb 1;<span class="ref-vol">192</span>(3):1294–301.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/24391218" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 24391218</span></a>] [<a href="http://dx.crossref.org/10.1093/glycob/cwn158" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="g98-recmanbinpro.REF.6">Ma Y, Shida H, Kawasaki T. Functional Expression of Human Mannan-Binding Proteins (MBPs) in Human Hepatoma Cell Lines Infected by Recombinant Vaccinia Virus: Post-Translational Modification, Molecular Assembly, and Differentiation of Serum and Liver MBP1. <span><span class="ref-journal">J Biochem. </span>1997;<span class="ref-vol">122</span>:810–8.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/9399586" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 9399586</span></a>] [<a href="http://dx.crossref.org/10.1093/oxfordjournals.jbchem.a021827" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK594022_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g98-recmanbinpro.FN1"><p class="no_top_margin">The authors declare no competing or financial interests.</p></div></dd></dl><div class="bk_prnt_sctn"><h2>Figures</h2><div class="whole_rhythm bk_prnt_obj bk_first_prnt_obj"><div id="g98-recmanbinpro.F1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%201%3A%20.%20Structural%20and%20functional%20expression%20of%20recombinant%20human%20mannan-binding%20protein%20(MBP).&p=BOOKS&id=594022_g98-recmanbinpro-Image001.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK594022/bin/g98-recmanbinpro-Image001.jpg" alt="Figure 1: . Structural and functional expression of recombinant human mannan-binding protein (MBP)." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>Structural and functional expression of recombinant human mannan-binding protein (MBP).</p><p>(C) Dose dependence of complement activation by peaks II–V eluted from the Sephacryl S-300 HR 10/100 column. Mannan-coated sheep erythrocytes are sensitized with 25–500 ng of peaks II–V and then lysed with complement (two volumes of CH50). ●, peak II; ○, peak III; ■, peak IV; and □, peak V.</p><p>(B) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of peaks II–V eluted from the Sephacryl S-300 HR 10/100 column. Samples of peaks II–V are electrophoresed on a 3%–10% polyacrylamide gel under nonreducing conditions. Recombinant human MBP produced in HLF cells exhibits similarities to the native human MBP. Positions of the marker proteins are indicated at the left. Lane 1, recombinant human MBP before gel filtration; lane 2, peak II; lane 3, peak III; lane 4, peak IV; and lane 5, peak V.</p><p>(A) Gel filtration chromatography of recombinant human MBP expressed in HLF hepatoma cells. Recombinant human MBP is analyzed by gel filtration chromatography on a Sephacryl S-300 HR 10/100 column. Elution positions of the marker proteins are indicated at the top, and numbers of the peaks are given above the peaks. BD, blue dextran (2,000 kDa); THY, thyroglobulin (669 kDa); ferritin (440 kDa); ALD, aldolase (158 kDa); BSA, bovine serum albumin (67 kDa).</p></div><div class="permissions">This figure was originally published in J Biochem. 122(4):810–8. 1997 "Functional expression of human mannan-binding proteins (MBPs) in human hepatoma cell lines infected by recombinant vaccinia virus: post-translational modification, molecular assembly, and differentiation of serum and liver MBP.” Ma Y. et al. Oxford University Press.</div></div></div></div><div id="bk_toc_contnr"></div></div></div>
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