Optimizing Recombinant Baculovirus Vector Design for Protein Production in Insect Cells

Carina Bannach,Adam C Chambers,Leo P Graves,Genna Bobby,Sainan Li,Raquel Arinto-Garcia,Linda A King,Robert D Possee,Daniel Ruiz Buck,Elizabeth Gan

doi:10.3390/pr9122118

Abstract

Autographa californica nucleopolyhedrovirus is a very productive expression vector for recombinant proteins in insect cells. Most vectors are based on the polyhedrin gene promoter, which comprises a TAAG transcription initiation motif flanked by 20 base pairs upstream and 47 base pairs downstream before the native ATG. Many transfer vectors also include a short sequence downstream of the ATG, in which case this sequence is mutated to ATT to abolish translation. However, the ATT sequence, or AUU in the mRNA, is known to be leaky. If a target-coding region is placed in the frame with the AUU, then some products will comprise a chimeric molecule with part of the polyhedrin protein. In this study, we showed that if AUU is placed in the frame with a Strep tag and eGFP coding region, we could identify a protein product with both sequences present. Further work examined if alternative codons in lieu of AUG might reduce translation initiation further. We found that AUA was used slightly more efficiently than AUU, whereas AUC was the least efficient at initiating translation. The use of this latter codon suggested that there might also be a slight improvement of protein yield if this is incorporated into expression vectors.

Highlights

Published: 25 November 2021One of the platforms predominantly used for eukaryotic recombinant protein synthesis is the baculovirus expression vector system (BEVS)
Protein Synthesis Is Initiated at AUU in Polh 50 UTR
We determined if translation could initiate from the AUU codon in the polh mRNA produced by many baculovirus expression vectors

Summary

Introduction

One of the platforms predominantly used for eukaryotic recombinant protein synthesis is the baculovirus expression vector system (BEVS). It utilises recombinant baculoviruses, mainly Autographa californica nucleopolyhedrovirus (AcMNPV) for high-level expression of foreign genes. Very high yields of functional proteins can be achieved, which undergo posttranslational modifications and processing. The manufacturing process is simple, fast, and can be scaled-up for insect cells. They can be used to transduce mammalian cells by expressing the foreign gene under the control of a mammalian-specific promoter.

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