to yeast, filamentous fungi can grow rapidly on inexpensive media, secrete large amounts of proteins, process eukaryotic mRNA, and carry out many posttranslational modifications. However, an additional advantage of using filamentous fungi as hosts for the production of mammalian proteins is their ability to add mammalian-like sugars to proteins.
Table 3.13 Some recombinant proteins produced by filamentous fungal expression systems
In sum, fungal expression systems play an important role in the production of heterologous proteins for research, industrial, and medical applications. However, experience has shown that no one system is able to produce an authentic version of every heterologous protein. For this and other reasons, gene expression systems that use insect or mammalian cells have been developed.
Baculovirus–Insect Cell Expression Systems
Baculoviruses are a large, diverse group of double-stranded DNA viruses that specifically infect arthropods, including many insect species, and are not infectious to other animals. During the infection cycle, two forms of baculovirus are produced (Fig. 3.34). Infection is initiated when the occluded form of the virus is ingested by the insect larvae. In this form, the viral nucleocapsids (virions) are clustered in a matrix that is made up of the protein polyhedrin, which protects the virions from degradation in the environment. The occluded virions packaged in this protein matrix are referred to as a polyhedron. Following ingestion, the virus is taken up into the midgut of the insect, the polyhedrin matrix dissolves due to the alkaline gut environment, and the virions enter midgut cells. The virions migrate to the nucleus where they are uncoated, releasing the DNA for genome replication, synthesis of viral proteins, and production of new virions. Within the insect midgut, the infection can spread from cell to cell as viral particles (single nucleocapsids) bud off from an infected cell and infect other midgut cells. This form of the virus, known as the budding form, is not embedded in a polyhedrin matrix and is not infectious to other individual insect hosts, although it can infect cultured insect cells. Plaques produced in insect cell cultures by the budding form of baculovirus have a different morphology from those produced by the occluded form. During the late stages of the infection cycle in the insect host, about 36 to 48 hours after infection, the polyhedrin protein is produced in massive quantities and continues for 4 to 5 days, until the infected cells rupture and the host organism dies. Occluded virions are released and can infect new hosts.
Figure 3.34 Budded (A) and occluded (B) forms of baculovirus. During budding, a nucleocapsid becomes enveloped by the membrane of an infected cell. A polyhedron consists of clusters of nucleocapsids (occluded virions) embedded in various orientations in a polyhedrin matrix.
The promoter for the polyhedrin (polyh) gene is exceptionally strong, and transcription from this promoter can account for as much as 25% of the mRNA produced in cells infected with the virus. Moreover, the polyhedrin protein is not required for virus production. Consequently, it was reasoned that replacement of the polyhedrin gene with a coding sequence for a heterologous protein, followed by infection of cultured insect cells, would result in the production of large amounts of the heterologous protein. Furthermore, because of the similarity of posttranslational modification systems between insects and mammals, it was thought that the recombinant protein would mimic closely, if not precisely, the authentic form of the original protein. Baculoviruses have been highly successful as delivery systems for introducing target genes for production of high levels of heterologous proteins in insect cells. More than a thousand different proteins have been produced using this system, including several vaccines that have been approved for veterinary or human use (Table 3.14).
Table 3.14 Some recombinant products produced by baculovirus-insect cell expression systems
The specific baculovirus that has been used extensively as an expression vector is Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). A. californica (the alfalfa looper) and over 30 other insect species are infected by AcMNPV. This virus also grows well on many insect cell lines. The most commonly used cell line for genetically engineered AcMNPV is derived from the fall armyworm, Spodoptera frugiperda. In these cells, the polyhedrin promoter is exceptionally active, and during infections with wild-type baculovirus, high levels of polyhedrin are synthesized.
Baculovirus Expression Vectors
The first step in the production of a recombinant AcMNPV that will be used to deliver the gene of interest into the insect host cell is to create a transfer vector. The transfer vector is an E. coli-based plasmid that carries a segment of DNA from AcMNPV (Fig. 3.35A) consisting of the polyhedrin promoter region with flanking upstream AcMNPV DNA, a multiple cloning site, the polyhedrin transcription termination and polyadenylation signal regions with flanking downstream AcMNPV DNA. The coding region for the polyhedrin gene has been deleted from this segment of DNA. The flanking AcMNPV DNA sequences provide regions for homologous recombination with the AcMNPV genome. A gene of interest is inserted into the multiple cloning site between the polyhedrin promoter and termination sequences, and the transfer vector is propagated in E. coli.
Figure 3.35 (A) Organization of the expression unit of a baculovirus (AcMNPV) transfer vector. The gene of interest is inserted into the multiple cloning site (MCS) that lies between the polyhedrin gene promoter (Pp) and polyhedrin gene transcription termination (Pt) sequences. The AcMNPV DNA upstream from the polyhedrin promoter (5′ AcMNPV DNA) and downstream from the polyhedrin transcription termination sequence (3′ AcMNPV DNA) provides sequences for integration of the expression unit by homologous recombination into an AcMNPV genome. (B) Replacement of the AcMNPV polyhedrin gene with an expression unit from a transfer vector. A double-crossover event (×) between homologous DNA segments of the transfer vector and the AcMNPV genome results in the integration of the expression unit into the AcMNPV genome. GOI, gene of interest.
Next, insect cells in culture are cotransfected with AcMNPV DNA and the transfer vector carrying the cloned gene. Within some of the doubly transfected cells, a double-crossover recombination event occurs at homologous sequences on the transfer vector and in the AcMNPV genome, and the cloned gene with polyhedrin promoter and termination regions becomes integrated into the AcMNPV DNA (Fig. 3.35B) with the concomitant loss of the polyhedrin gene. Virions lacking the polyhedrin gene produce distinctive zones of cell lysis (occlusion-negative plaques), from which recombinant baculovirus is isolated.
Linearization of the AcMNPV genome before transfection into insect cells substantially increases the frequency of recombinant plaques. The AcMNPV genome was engineered with two Bsu36I sites that were placed on either side of the polyhedrin gene (Fig. 3.36). One is in gene 603 and the other is in gene 1629 that is essential for viral replication. When DNA from this modified baculovirus is treated with Bsu36I and transfected into insect cells, no viral replication occurs because a segment of the essential gene (1629) is missing. As part of this system, a transfer vector is constructed with the gene of interest between intact versions of gene 603 and gene 1629. This transfer vector is introduced into insect cells that were previously transfected with linearized, replication-defective AcMNPV DNA that is missing the segment between the two Bsu36I sites. A double-crossover event both reestablishes a functional version of gene 1629 and incorporates the cloned gene into the AcMNPV genome (
