A Guide to Successful Scale-up of the Baculovirus Expression System

Mannix, C.J.; Jarman, Rick

doi:10.1007/978-94-011-4315-8_3

Cited by 6 publications

(5 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The most common approaches for expressing enzymatically active recombinant human DPP4 uses baculovirus-infected insect cells [11,12,32,33], and less commonly mammalian and yeast cells [9,34]. Baculovirus-insect expression systems have a number of advantages over mammalian and yeast expression systems, including the ease of use, adequate glycosylation, appropriate codon usage and potentially greater expression [35,36]. Although glycosylation of recombinant proteins in insect cells is less complex than the native human glycoproteins, it has produced stable and active soluble recombinant human DPP4 proteins [11].…”

Section: Introductionmentioning

confidence: 99%

A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

Xi¹,

Fazio²,

Nadvi³

et al. 2020

Preprint

View full text Add to dashboard Cite

Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulfate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor binding domain (RBD) were measured using surface plasmon resonance. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.

show abstract

Section: Introductionmentioning

confidence: 99%

A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

Xi¹,

Fazio²,

Nadvi³

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…Additionally, the baculovirus is genetically unstable and it may be necessary to reduce the amount of baculovirus used in production by designing a single baculovirus which bears all the genetic elements required . For further information on baculovirus biology and its application in biopharmaceutical research and production the reader is referred to the excellent reviews by Kost et al (2000) and Mannix and Jarman (2000).…”

Section: Adeno-associated Viral Vectorsmentioning

confidence: 99%

Cell Culture Processes for the Production of Viral Vectors for Gene Therapy Purposes

2006

View full text Add to dashboard Cite

Gene therapy is a promising technology for the treatment of several acquired and inherited diseases. However, for gene therapy to be a commercial and clinical success, scalable cell culture processes must be in place to produce the required amount of viral vectors to meet market demand. Each type of vector has its own distinct characteristics and consequently its own challenges for production. This article reviews the current technology that has been developed for the efficient, large-scale manufacture of retrovirus, lentivirus, adenovirus, adeno-associated virus and herpes simplex virus vectors.

show abstract

“…All cultures were sampled as indicated. The titers of the BacMam viruses were determined as previously described (Mannix 2000) using Sf9 cells.…”

Section: Expression Of Saf-3-fc In Cho and Cos-7 Cells Infected With Recombinant Baculovirusmentioning

confidence: 99%

Untitled

et al. 2002

View full text Add to dashboard Cite

Baculovirus containing the mammalianCMV promoter, in place of the insect polyhedronpromoter (BacMam), has been used to transientlytransfect COS, CHO and CHOE1a (CHO cells expressing theE1a transcriptional activator). Using this system forthe expression of a cellular adhesion factor (SAF-3) Fcfusion protein in CHOE1a, we found that levels ofexpression were highest with a MOI of 100, 20mM sodiumbutyrate, at 34 degrees C. Production increased furtherif the cells were resuspended in fresh medium, about3 x 10(6) cells ml(-1), prior to addition of the virus. These conditions were used to express 3 secretedproteins, SAF-3-Fc, CD40-hexa his and Asp 2-Fc, and, at2 to 6 days post infection, protein levels ranged from4 ug ml(-1) to 25 ug ml(-1). Based on these results, theBacMam system represents a viable technique forproducing protein at ug ml(-1) levels in a relatively shortperiod of time.

show abstract

A Guide to Successful Scale-up of the Baculovirus Expression System

Cited by 6 publications

References 32 publications

A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

Cell Culture Processes for the Production of Viral Vectors for Gene Therapy Purposes

Untitled

Contact Info

Product

Resources

About