Mass spectrometry analysis reveals differences in the host cell protein species found in pseudotyped lentiviral vectors
Autor: | Yasuhiro Takeuchi, Robin Thorpe, Yuan Zhao, Mary Collins, Jun X. Wheeler, Sabine Johnson |
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Rok vydání: | 2018 |
Předmět: |
0301 basic medicine
Genetic enhancement Genetic Vectors Endogenous retrovirus Host proteins Bioengineering Tandem mass spectrometry Applied Microbiology and Biotechnology Article Virus Small hairpin RNA 03 medical and health sciences 0302 clinical medicine ALIX Animals Humans TSG101 Vector (molecular biology) Virus Release Pharmacology Gene knockdown Mass spectrometry General Immunology and Microbiology Chemistry Virus Assembly Lentivirus Lentiviral vectors General Medicine AHNAK Cell biology HEK293 Cells 030104 developmental biology 030220 oncology & carcinogenesis Cats Biotechnology |
Zdroj: | Biologicals |
ISSN: | 1045-1056 |
Popis: | Lentiviral vectors (LVs) have been successfully used in clinical trials showing long term therapeutic benefits. Studying the role of cellular proteins in lentivirus HIV-1 life cycle can help understand virus assembly and budding, leading to improvement of LV production for gene therapy. Lentiviral vectors were purified using size exclusion chromatography (SEC). The cellular protein composition of LVs produced by two different methods was compared: the transient transfection system pseudotyped with the VSV-G envelope, currently used in clinical trials, and a stable producer cell system using a non-toxic envelope derived from cat endogenous retrovirus RD114, RDpro. Proteins of LVs purified by size exclusion chromatography were identified by tandem mass spectrometry (MS/MS). A smaller number of cellular protein species were detected in stably produced vectors compared to transiently produced vector samples. This may be due to the presence of co-purified VSV-G vesicles in transiently produced vectors. AHNAK (Desmoyokin) was unique to RDpro-Env vectors. The potential role in LV particle production of selected proteins identified by MS analysis including AHNAK was assessed using shRNA gene knockdown technique. Down-regulation of the selected host proteins AHNAK, ALIX, and TSG101 in vector producer cells did not result in a significant difference in vector production. |
Databáze: | OpenAIRE |
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