InsectSelect™ System From Invitrogen

Production of recombinant proteins is an essential part of research in laboratories today. Many labs use baculovirus expression systems to express their proteins in insect cells. While protein yields in these systems are good, making the recombinant baculoviruses is a time and resource hungry process. Invitrogen has developed the Insect Select™ System which uses elements of the baculovirus expression system to express proteins, without needing to produce the baculovirus itself.

The basic kit contains a cloning vector in three reading frames for easy cloning, a control expression plasmid (CAT) and sequencing primers for sequence analysis of recombinant expression vectors. Note that none of the products required for insect cell expression are included.

The new technology uses a single expression vector, and there are several available from Invitrogen that have options for secreted or non-secreted proteins as well as a choice of antibiotic. The basic vector (pIB/V5-His) contains baculovirus promotors (OpIE2, OpIE1) which do not require viral activation for expression in insect cells. The vector contains the ampicillin resistance gene for selection in bacteria and a second antibiotic resistance gene (e.g. blasticidin) is included and under the control of a baculovirus promotor so that it can be used to select stable insect cell lines expressing your protein. The vector also contains a C-terminal peptide which contains a polyhistidine tag and a V5 epitope for ease of purification and detection. Vector options include pMIB/V5-His which contains a honeybee melittin secretion signal and pIZT/V5-His which contains a GFP/Zeocin™ fusion gene that allows visual monitoring of transfection efficiency.

Getting to a usable expression vector is reasonably straight-forward: Clone your gene of interest into the vector and check by sequence and restriction analysis. The next step is to transfect the vector into insect cells and check for transient protein expression. If desired, a stable insect cell line can be selected using blasticidin or Zeocin™ and protein production scaled up. Invitrogen states that selection of a stable cell line can take as little as two weeks if high levels of antibiotic are used.

In our laboratory, we have trialed this system alongside traditional baculovirus methods. As with the Bac-to-Bac® Baculovirus Expression System, some skill with insect cell culture is required for rapid success as insect cells can be sensitive to minor changes in culture conditions. The time from cloning to protein expression is much faster using the InsectSelect™ System; however, if a stable cell line is required, the cost of continuous antibiotic selection should be considered, especially if cells are to be maintained constantly. (Large cultures for protein production do not necessarily need antibiotic present.) Invitrogen also admit that while the OpIE2 promotor enables reasonable levels of expression, it may not be as high as baculovirus-driven expression. It is essential to put down healthy liquid nitrogen stocks of low-passage cell lines to prevent loss of the cell line – refer to the cell culture guide from Invitrogen for more information.