title: Enhanced expression of recombinant proteins utilizing a modified baculovirus expression vector.
abstract: The baculovirus expression vector system (BEVS) has been widely used for over-expressing eukaryotic proteins due to a close resemblance in post-translational modification, processing, and transportation properties of the expressed protein, to that of the mammalian cells. In comparison to the bacterial expression system, protein yield from BEVS is relatively low, resulting in higher cost of production. To improve the existing recombinant protein expression levels, baculovirus homologous region1 (hr1) was strategically integrated into the bacmid-based transfer vectors. Luciferase reporter, human Protein Kinase B-alpha (PKB-A), and N-terminal-modified CYP-1A2 genes were independently cloned in non-hr1 and hr1 constructs for generating respective bacmids and baculoviruses. These recombinant baculoviruses were utilized for comparing the expression levels at varying multiplicity of infections (MOI) and time intervals in Spodoptera frugiperda (Sf21) or Trichoplusia ni (Tni) insect cell lines. Targeted insertion of hr1 upstream to CYP-1A2, PKB-A, and Luciferase genes, compared to the non-hr1 sets, led to 3-, 3.5-, and 4.5-fold increase in the resultant protein levels, respectively. Moreover, at equal protein concentration, the corresponding activity and inhibition characteristics of these high expression hr1 sets were comparable to that of the respective non-hr1 sets. Utilization of this modified baculovirus expression construct offers significant advantage of producing recombinant proteins in a cost-effective manner for various biotechnological and therapeutic applications.
authors.affiliation: Department of Molecular Technology, New Drug Discovery Research, Ranbaxy Research Laboratories-R&D-3, 20-Sector 18 Udyog Vihar, Gurgaon, India. prabhakar.tiwari@ranbaxy.com
authors.forename: Prabhakar
authors.initials: P
authors.lastname: Tiwari
authors.forename: Shalini
authors.initials: S
authors.lastname: Saini
authors.forename: Sanatan
authors.initials: S
authors.lastname: Upmanyu
authors.forename: Biju
authors.initials: B
authors.lastname: Benjamin
authors.forename: Ruchi
authors.initials: R
authors.lastname: Tandon
authors.forename: Kulvinder Singh
authors.initials: KS
authors.lastname: Saini
authors.forename: Sudhir
authors.initials: S
authors.lastname: Sahdev
mesh_terms: D016367:Baculoviridae / Q000235:genetics*; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D008156:Luciferases; D019388:Cytochrome P-450 CYP1A2; D051057:Proto-Oncogene Proteins c-akt