1-3.10.2 Baculoviral Vectors:

Baculovirus is a DNA virus with host range restricted to invertebrates, mostly insects. The baculovirus expression system has been used extensively for the expression of recombinant proteins in insect cells. Baculovirus is a group of insect virus and Autographa californica nucleopolyhedrovirus (AcMNPV) is the most extensively studied virus under this family.

The infection of AcMNPV is initiated by replication and transcription of of the DNA genome inside the nucleus and the assembly of the nucleocapsids. The nucleocapsids then bud off from the plasma membrane and initiate systemic infection.

Although, baculoviral vectors can transfect only insect cells, recombinant baculoviral vectors have been constructed containing mammalian cell specific promoters which can be used to infect mammalian cells as well.

Advantages of baculoviral vectors:

•  Since insects cells are high eukaryotes, desired port-translational modification of complex protein can be achieved.

•  They have higher packaging capacity of insert.

•  Lower biosafety issue.

•  High level of protein expression.

There are few drawbacks of baculoviral vectors such as-

•  Foreign protein expression using an insect system is more complex and time consuming than a bacterial system.

•  Sometime protein post-translational processing may be sub-optimal to compensate the secretory pathway of the protein.

1-3.11 Yeast Vector System:

Cloning and expression of a gene using yeast system has several advantages over E.coli system due to presence of eukaryotic post-translational modification machinery. Expression of complex proteins with proper modification and folding can be achieved by yeast eukaryotic system. These vectors have yeast origin of replication (ARS) for replication and maintenance in the yeast system and bacterial ori for maintaining inside a bacterial system.

Different types of yeast vector include YIp (yeast integrative plasmid), YEp (yeast episomal plasmid), YRp (yeast replicating plasmid), YCp (yeast centromere plasmid) etc.

YIp (yeast integrative plasmid) can integrate to the host genome by homologous recombination. This generally yields a single copy of recombinant vector DNA integrated to the host genome.

YEp (yeast episomal plasmid) can be maintained in the yeast system as an autonomously replicating episomal plasmid. This vector contains a part of 2μ plasmid which is essential for autonomous replication of the vector inside the yeast.

YRp (yeast replicating plasmid) are used to obtain a high copy number inside the host (upto 100 copy number).

YCp (yeast centromere plasmid) possess a centromeric region in addition to ARS which facilitates the mitotic segregation of the linear plasmid during replication. The copy number of this vector is essentially one per cell.