title: Single-step concentration and purification of adenoviruses by coxsackievirus-adenovirus receptor-binding capture and elastin-like polypeptide-mediated precipitation.
abstract: A single-step method for quick concentration and purification of adenoviruses (Ads) was established by combining coxsackievirus and adenovirus receptor (CAR)-binding capture with elastin-like polypeptide (ELP)-mediated precipitation. The soluble ELP-CAR fusion protein was expressed in vector-transformed E. coli and purified to high purity by two rounds of inverse transition cycling (ITC). After demonstration of the specific binding of fusion protein, a recombinant Ad (rAd), namely rAd/GFP, was pulled down from the culture medium and extract of rAd-transduced cells using ELP-CAR protein, with recovery of 76.2 % and 73.3 %, respectively. The rAd was eluted from the ELP-CAR protein and harvested by one round of ITC, with recoveries ranging from 30.6 % to 34.5 % (virus titration assay). Both ELP-CAR-bound and eluted rAds were able to transduce CAR-positive cells, but not CAR-negative cells (fluorescent microscopy). A further viral titration assay showed that the ELP-CAR-bound rAd/GFP had significantly lower transduction efficiency than the eluted rAd, and there was less of a decrease when tested in the presence of fetal bovine serum. In addition, rAd/GFP was efficiently recovered from the "spiked" PBS and tap water with recovery of ~74 % or ~60 %. This work demonstrates the usefulness of the ELP-CAR-binding capture method for concentration and/or purification of Ads in cellular and environmental samples.
authors.affiliation: College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal infectious Diseases and Zoonoses, Yangzhou University, 48 Wenhui Road, Yangzhou, 225009, Jiangsu, People's Republic of China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Wu
authors.affiliation: College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal infectious Diseases and Zoonoses, Yangzhou University, 48 Wenhui Road, Yangzhou, 225009, Jiangsu, People's Republic of China.
authors.forename: Wenjun
authors.initials: W
authors.lastname: Liu
authors.affiliation: College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal infectious Diseases and Zoonoses, Yangzhou University, 48 Wenhui Road, Yangzhou, 225009, Jiangsu, People's Republic of China.
authors.forename: Bi
authors.initials: B
authors.lastname: Xu
authors.affiliation: College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal infectious Diseases and Zoonoses, Yangzhou University, 48 Wenhui Road, Yangzhou, 225009, Jiangsu, People's Republic of China.
authors.forename: Xinyu
authors.initials: X
authors.lastname: Zhang
authors.affiliation: College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal infectious Diseases and Zoonoses, Yangzhou University, 48 Wenhui Road, Yangzhou, 225009, Jiangsu, People's Republic of China.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Xia
authors.affiliation: College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal infectious Diseases and Zoonoses, Yangzhou University, 48 Wenhui Road, Yangzhou, 225009, Jiangsu, People's Republic of China. sunh@yzu.edu.cn.
authors.forename: Huaichang
authors.initials: H
authors.lastname: Sun
mesh_terms: D000256:Adenoviridae / Q000302:isolation & purification*; D000818:Animals; D002460:Cell Line; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D004549:Elastin / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013552:Swine; D014161:Transduction, Genetic; D014773:Virology / Q000379:methods*
chemical_list: D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D011993:Recombinant Fusion Proteins; D004549:Elastin