Abstract

The baculovirus expression vector system (BEVS) is becoming the method of choice for expression of many eukaryotic proteins and protein complexes for biochemical, structural and pharmaceutical studies. Significant technological advancement has made generation of recombinant baculoviruses easy, efficient and user-friendly. However, there is a tremendous variability in the amount of proteins made using the BEVS, including different batches of virus made to express the same proteins. Yet, what influences the overall production of proteins or protein complexes remains largely unclear. Many downstream applications, particularly protein structure determination, require purification of large quantities of proteins in a repetitive manner, calling for a reliable experimental set-up to obtain proteins or protein complexes of interest consistently. During our investigation of optimizing the expression of the Mediator Head module, we discovered that the ‘initial infectivity’ was an excellent indicator of overall production of protein complexes. Further, we show that this initial infectivity can be mathematically described as a function of multiplicity of infection (MOI), correlating recombinant protein yield and virus titer. All these findings led us to develop the Titer Estimation for Quality Control (TEQC) method, which enables researchers to estimate initial infectivity, titer/MOI values in a simple and affordable way, and to use these values to quantitatively optimize protein expressions utilizing BEVS in a highly reproducible fashion.

Highlights

  • The baculovirus expression vector system (BEVS), introduced about 30 years ago [1,2,3,4,5], has become an essential tool for expression of many eukaryotic proteins and protein complexes in insect cells [6,7,8]

  • The initial infectivity strongly correlates with overall protein complex production In our previous work, we used the MultiBac baculovirus expression system as our method of choice for high-yield, recombinant protein production [16]: As illustrated in Fig 1A, the multigene construct harboring all 7 genes encoding the Mediator Head subunits was generated, and integrated into baculovirus genome (DH10MultiBac) followed by virus production, expression and purification of the complex [16] (Fig 1A)

  • This work has revealed the connection among initial infectivity, multiplicity of infection (MOI), and overall recombinant protein production

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Summary

Introduction

The baculovirus expression vector system (BEVS), introduced about 30 years ago [1,2,3,4,5], has become an essential tool for expression of many eukaryotic proteins and protein complexes in insect cells [6,7,8]. BEVS has been proven powerful for structure determination of membrane. TECQ method for optimal expressions of protein complexes using the BEVS

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