{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,7]],"date-time":"2026-09-07T03:26:59Z","timestamp":1788751619875,"version":"build-2803163510"},"reference-count":43,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2022,3,10]],"date-time":"2022-03-10T00:00:00Z","timestamp":1646870400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100010245","name":"Social Science Foundation of Liaoning Province","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100010245","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Mol. Biosci."],"abstract":"<jats:p>Antigen-binding variable domains of the H chain of heavy-chain antibodies (VHHs), also known as nanobodies (Nbs), are of great interest in imaging technique, disease prevention, diagnosis, and therapy. High-level expression of soluble Nbs is very important for its industrial production. In this study, we optimized the expression system of anti-green fluorescent protein (GFP) VHHs with three different signal peptides (SPs), outer-membrane protein A (OmpA), pectate lyase B (PelB), and L-asparaginase II SP (L-AsPsII), in different <jats:italic>Escherichia coli<\/jats:italic> strains <jats:italic>via<\/jats:italic> isopropyl \u03b2-D-thiogalactoside (IPTG) induction and auto-induction, respectively. The solubility of recombinant anti-GFP VHHs with PelB or OmpA was significantly enhanced to the same extent by IPTG induction and auto-induction in BL21 (DE3) <jats:italic>E. coli<\/jats:italic> strain and the maximum yield of target protein reached approximately 0.4\u00a0mg\/l in a shake flask. The binding activity of recombinant anti-GFP VHHs was also confirmed to be retained by native-polyacrylamide gel electrophoresis (PAGE). These results suggest that SPs like OmpA and PelB could efficiently improve the recombinant anti-GFP VHH solubility without changing its bioactivity, providing a novel strategy to optimize the <jats:italic>E. coli<\/jats:italic> expression system of soluble VHHs, and lay the foundation for the industrial production of soluble recombinant anti-GFP VHHs and the research of other VHHs in the future.<\/jats:p>","DOI":"10.3389\/fmolb.2022.848829","type":"journal-article","created":{"date-parts":[[2022,3,10]],"date-time":"2022-03-10T12:27:39Z","timestamp":1646915259000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":15,"title":["Highly Expressed Soluble Recombinant Anti-GFP VHHs in Escherichia coli via Optimized Signal Peptides, Strains, and Inducers"],"prefix":"10.3389","volume":"9","author":[{"given":"Shuangying","family":"Chao","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuhang","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ning","family":"Ding","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yue","family":"Lin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qian","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junwen","family":"Tan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yang","family":"Zheng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xuejun","family":"Hu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junming","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2022,3,10]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"393","DOI":"10.1016\/j.jmb.2006.07.034","article-title":"Systematic Screening of All Signal Peptides from Bacillus Subtilis: a Powerful Strategy in Optimizing Heterologous Protein Secretion in Gram-Positive Bacteria","volume":"362","author":"Brockmeier","year":"2006","journal-title":"J. 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