Enhanced expression of recombinant proteins utilizing a modified baculovirus expression vector |
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Authors: | Prabhakar Tiwari Shalini Saini Sanatan Upmanyu Biju Benjamin Ruchi Tandon Kulvinder Singh Saini Sudhir Sahdev |
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Institution: | (1) Department of Molecular Technology, New Drug Discovery Research, Ranbaxy Research Laboratories-R&D-3, 20-Sector 18 Udyog Vihar, Gurgaon, 122001, India;(2) Department of Metabolism and Pharmacokinetics, New Drug Discovery Research, Ranbaxy Research Laboratories, Gurgaon, India;(3) Department of Pharmacology, New Drug Discovery Research, Ranbaxy Research Laboratories, Gurgaon, India |
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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-α (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 expresion 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. |
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