Selection of novel vesicular stomatitis virus glycoprotein variants from a peptide insertion library for enhanced purification of retroviral and lentiviral vectors

Journal of Virology
Julie H Yu, David V Schaffer

Abstract

The introduction of new features or functions that are not present in an original protein is a significant challenge in protein engineering. For example, modifications to vesicular stomatitis virus glycoprotein (VSV-G), which is commonly used to pseudotype retroviral and lentiviral vectors for gene delivery, have been hindered by a lack of structural knowledge of the protein. We have developed a transposon-based approach that randomly incorporates designed polypeptides throughout a protein to generate saturated insertion libraries and a subsequent high-throughput selection process in mammalian cells that enables the identification of optimal insertion sites for a novel designed functionality. This method was applied to VSV-G in order to construct a comprehensive library of mutants whose combined members have a His6 tag inserted at likely every site in the original protein sequence. Selecting the library via iterative retroviral infections of mammalian cells led to the identification of several VSV-G-His6 variants that were able to package high-titer viral vectors and could be purified by Ni-nitrilotriacetic acid affinity chromatography. Column purification of vectors reduced protein and DNA impurities more than 5,000-fold and 1...Continue Reading

References

Jun 15, 1992·Proceedings of the National Academy of Sciences of the United States of America·M Gossen, H Bujard
Jan 26, 1987·Nucleic Acids Research·B LuckowG Schütz
Oct 25, 1994·Proceedings of the National Academy of Sciences of the United States of America·W P Stemmer
Sep 1, 1996·Journal of Virology·J M Le DouxM L Yarmush
Oct 15, 1996·Proceedings of the National Academy of Sciences of the United States of America·L NaldiniI M Verma
Feb 18, 1997·Proceedings of the National Academy of Sciences of the United States of America·I R SinghP O Brown
Jun 1, 1997·Nature Biotechnology·E T Boder, K D Wittrup
Jul 22, 1997·Proceedings of the National Academy of Sciences of the United States of America·H Zhao, F H Arnold
Sep 4, 1997·Nature·J F PresleyJ Lippincott-Schwartz
Sep 12, 1998·Journal of Virology·H MiyoshiI M Verma
Oct 10, 1998·Journal of Virology·T DullL Naldini
Dec 16, 1998·Journal of Virology·T KafriI M Verma
Feb 28, 2002·Molecular Therapy : the Journal of the American Society of Gene Therapy·Keith Bupp, Monica J Roth
Apr 27, 2002·Human Gene Therapy·Veerle BaekelandtZeger Debyser
May 30, 2002·Nucleic Acids Research·Yaron S N ButterfieldSteven J M Jones
Dec 7, 2002·Nature Neuroscience·Karen LaiDavid V Schaffer
Dec 13, 2002·Journal of Virology·Heikki VilenHarri Savilahti
May 27, 2003·BioTechniques·Kaoru YamadaChristopher E Walsh
Sep 25, 2003·Proceedings of the National Academy of Sciences of the United States of America·Marcy R AuerbachIla R Singh
Jan 27, 2004·Journal of Molecular Biology·Gurkan Guntas, Marc Ostermeier
Jan 27, 2004·Molecular Therapy : the Journal of the American Society of Gene Therapy·Ghiabe H GuibingaTheodore Friedmann
May 27, 2004·Biotechnology and Bioengineering·Jolene M Ignowski, David V Schaffer
Apr 7, 2005·Biotechnology and Bioengineering·María de las Mercedes SeguraAlain Garnier
Jun 21, 2005·Molecular Therapy : the Journal of the American Society of Gene Therapy·Peter J KretschmerTerry W Hermiston
Sep 9, 2005·Microbiology and Molecular Biology Reviews : MMBR·Ling YuanRobert Keenan

❮ Previous
Next ❯

Citations

Sep 18, 2007·Molecular Biotechnology·Adam S Cockrell, Tal Kafri
Jan 13, 2011·Molecular Therapy : the Journal of the American Society of Gene Therapy·Jae-Hyung JangDavid V Schaffer
Nov 24, 2011·Molecular Therapy : the Journal of the American Society of Gene Therapy·Prashanth AsuriDavid V Schaffer
Sep 18, 2012·Nature Neuroscience·Randolph S AshtonDavid V Schaffer
Jul 24, 2008·Annual Review of Biomedical Engineering·David V SchafferKwang-il Lim
Sep 5, 2006·Virology Journal·Hanna Dreja, Marc Piechaczyk
Jun 2, 2014·Nature Methods·Alex J HughesAmy E Herr
Oct 4, 2013·Journal of Virology·Arun AmmayappanStephen J Russell
Apr 18, 2013·Expert Opinion on Biological Therapy·Maria Mercedes SeguraAlain Garnier
Jul 1, 2015·Biologicals : Journal of the International Association of Biological Standardization·Jitendra K BiswalBramhadev Pattnaik
Jul 30, 2014·Biotechnology and Bioengineering·Ashley L FritzDavid V Schaffer
Feb 11, 2015·Molecular Therapy : the Journal of the American Society of Gene Therapy·Ashley L FritzDavid V Schaffer
Apr 23, 2015·Nature Communications·L J BugajD V Schaffer
Nov 11, 2008·Cell Host & Microbe·Joshua N LeonardDavid V Schaffer
Jul 20, 2007·Developmental Neurobiology·Joseph PeltierDavid V Schaffer
Jul 7, 2007·Biotechnology and Bioengineering·Jae-Hyung JangDavid V Schaffer
Feb 6, 2021·BMC Biotechnology·Shiaki A Minami, Priya S Shah

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.