High-Throughput Production of Proteins in E. coli for Structural Studies.

We have developed a standardized and efficient workflow for high-throughput (HT) protein expression in E. coli and parallel purification which can be tailored to the downstream application of the target proteins. It includes a one-step purification for the purposes of functional assays and a two-step protocol for crystallographic studies, with the option of on-column tag removal.

BacMam Delivery of a Protective Gene to Reduce Renal Ischemia-Reperfusion Injury.

Ischemia-reperfusion (I/R) injury remains the primary contributor to delayed graft function in kidney transplantation. The beneficial application of manganese superoxide dismutase (sod), delivered by a BacMam vector, against renal I/R injury has not been evaluated previously. Therefore, this study overexpressed sod-2 in proximal tubular epithelial (HK-2) cells and porcine kidney organs during simulated renal I/R injury.

High-throughput baculovirus expression in insect cells.

Historically, it has been proved difficult to adapt the traditional baculovirus expression systems to an automated platform because of the complexity of the processes involved. One of the major bottlenecks is the selection of recombinant from parental viruses. We have developed a bacmid vector (flashBAC™) that does not require any form of selection pressure to separate recombinant virus from nonrecombinant parental virus.

Optimizing the baculovirus expression vector system.

Baculoviruses have a unique bi-phasic life cycle and powerful promoters, which greatly facilitates their use for recombinant protein expression in insect cells. We have developed an expression system that utilizes homologous recombination in insect cells between a transfer plasmid containing a gene to be expressed and a replication-deficient virus (bacmid). Only recombinant virus can replicate facilitating the rapid production of multiple recombinant viruses using robotic liquid handlers.

Baculovirus as vectors for human cells and applications in organ transplantation.

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is able to transduce a wide range of mammalian cells and shows preferential uptake in some, particularly liver and kidney cells. This suggests that the virus may be useful for delivery of protective genes for ameliorating the effects of ischaemia reperfusion injury (IRI) in solid organs during transplantation procedures. In this chapter we discuss the advantages of the baculovirus over other virus vectors for gene delivery in organ transplantation and describe some of the protective genes which may be used to ameliorate the effects of IRI.

Baculovirus as delivery system for gene transfer during hypothermic organ preservation.

Concerns over the safety of conventional viral vectors have limited the translation of gene transfer from an exciting experimental procedure to a successful clinical therapy in transplantation. Baculoviruses are insect viruses, but have the ability to enter mammalian cells and deliver potential therapeutic molecules with no evidence of viral replication. This study provides evidence of the ability of recombinant baculovirus to enter mammalian kidneys and livers during cold preservation.

Improved expression of secreted and membrane-targeted proteins in insect cells.

Secretory and membrane-bound proteins are generally produced in lower amounts in insect cells compared with cytoplasmic and nuclear proteins. There may be many reasons for this, including degradation of recombinant proteins by proteases, competition for cellular resources between native and recombinant proteins, and physical blockage of the secretory pathways. In the present study, we describe the construction of a baculovirus in which chiA (chitinase) and cath (cathepsin) genes have been deleted and show improved recombinant protein expression using this vector.

Genetic modification of a baculovirus vector for increased expression in insect cells.

Generating large amounts of recombinant protein in transgenic animals is often challenging and has a number of drawbacks compared to cell culture systems. The baculovirus expression vector system (BEVS) uses virus-infected insect cells to produce recombinant proteins to high levels, and these are usually processed in a similar way to the native protein. Interestingly, since the development of the BEVS, the virus most often used (Autographa californica multi-nucleopolyhedovirus; AcMNPV) has been little altered genetically from its wild-type parental virus.

Baculovirus expression systems for recombinant protein production in insect cells.

Baculoviruses are lethal pathogens of insects, predominantly of the order Lepidoptera. These viruses have a bi-phasic life cycle, which greatly facilitates their use for biotechnological applications. They were exploited initially as biocontrol agents, and then engineered as protein expression vectors.

Generation of baculovirus vectors for the high-throughput production of proteins in insect cells.

The baculovirus expression system is one of the most popular methods used for the production of recombinant proteins but has several complex steps which have proved inherently difficult to adapt to a multi-parallel process. We have developed a bacmid vector that does not require any form of selection pressure to separate recombinant virus from non-recombinant parental virus. The method relies on homologous recombination in insect cells between a transfer vector containing a gene to be expressed and a replication-deficient bacmid.

Host mediated selection of pathogen genotypes as a mechanism for the maintenance of baculovirus diversity in the field.

The genetic diversity of many DNA virus populations in nature is unknown, but for those that have been studied it has been found to be relatively high. This is particularly true for baculoviruses, a family of large double-stranded DNA viruses that infect the larval stages of insects. Why there should be such heterogeneity within these virus populations is puzzling and what sustains it is still unknown.

Quantitative real-time PCR for rapid and accurate titration of recombinant baculovirus particles.

We describe the use of quantitative PCR (QPCR) to titer recombinant baculoviruses. Custom primers and probe were designed to gp64 and used to calculate a standard curve of QPCR derived titers from dilutions of a previously titrated baculovirus stock. Each dilution was titrated by both plaque assay and QPCR, producing a consistent and reproducible inverse relationship between C(T) and plaque forming units per milliliter.