Abstract
Baculovirus-insect cell expression system (BEVS) is one of the most popular methods used for the production of recombinant proteins owing to its capacity to produce many recombinant proteins at high yield and with functional activities. However, lower yields of secreted or membrane-bound proteins are usually obtained from insect cells than that of cytosol or nucleus proteins. In Chapters 2 and 3 I utilized a SEAP-eGFP (SEFP) fusion protein as a model reporter to identify potential genes that are able to elevate the production of secreted protein, SEFP, either from translational or transcriptional regulation. A series of bicistronic baculovirus vectors were constructed to co-express potential genes with SEFP in insect cells. Among them were (i) translation initiation factor, eIF4E; (ii) ER chaperone, calreticulin (CALR); and (iii) sHSP-like proteins, α-synuclein (α-syn) and β-synuclein (β-syn). SEAP enzymatic analysis and Western blot were carried out for comparison of enhanced extracellular proteins by candidate proteins with that of a negative control, Hsp40-like protein. In addition, expression of SEFP fusion protein in recombinant viruses provides a rapid method to trace the morphology of the special ring shape of SEFP under a fluorescent microscope. The mRNA expression level from each recombinant virus-infected cell was further analyzed by quantitative PCR. In all instances, cells co-expressed with α- and β-synuclein showed a greatly enhanced level of SEFP production and this might come from transcriptional up-regulation. A comparable SEFP production in cells co-expressed eIF4E was obtained when compared with that of the CALR co-expressed cells, although that was governed by a different mechanism. Finally, I co-expressed β-syn, eIF4E and CALR together with SEFP using a quadruple vector, pAcAB4. Not only an additive effect on SEFP production was shown, but also a prolonged life span in the infected Sf9 and 21 cell lines. Propidium iodide staining and Western blot were conducted for demonstrating the compactness of the infected cell membranes. In this thesis, it is clearly showed that the co-expression of chaperones and translation initiation factor facilitate production of secreted protein with a significantly delayed cell lysis. In other words, this quadruple baculovirus vector provides a longer time for protein processing in infected cells and will certainly facilitate glycol-protein production, structural protein research, and vaccine development for future applications.