The Production of Multiprotein Complexes in Insect Cells Using the Baculovirus Expression System

Abstract

The production of a homogeneous protein sample in sufficient quantities is an essential prerequisite not only for structural investigations but represents also a rate-limiting step for many functional studies. In the cell, a large fraction of eukaryotic proteins exists as large multicomponent assemblies with many subunits, which act in concert to catalyze specific activities. Many of these complexes cannot be obtained from endogenous source material, so recombinant expression and reconstitution are then required to overcome this bottleneck. This chapter describes current strategies and protocols for the efficient production of multiprotein complexes in large quantities and of high quality, using the baculovirus/insect cell expression system.

In TFIIH the Arch domain of XPD is mechanistically essential for transcription and DNA repair

“…Baculoviruses, protein production, and purification. The cDNAs encoding human MAT1 and XPD-FLAG tag were inserted into the transfer vector pAC8_MF under the control of its PH promoter and recombined with baculovirus DNA (Bac10:KO1629 Δ v-cath-Chia) in Sf9 cells to generate the corresponding viruses (V_MAT1 and V_XPD) as described previously 42 . Mouse anti-XPD(2F6), p44(1H5), MAT1(2F5), DsRed, and rabbit anti-MAT1 antibodies were obtained from IGBMC's facilities.…”

An evolutionary conserved Hexim1 peptide binds to the Cdk9 catalytic site to inhibit P-TEFb

“…The resulting protein was purified further on a heparin column eluted with a 0-350 mM NaCl gradient in 20 mM Hepes pH 7.2 and 2 mM DTT. To obtain P-TEFb, Cdk9 cDNA fused to an N-terminal strep tag and full-length cyclin T1 cDNA were cloned into a PKL MultiBac vector, under the control of polyhedrin and p10 promoters (51). Sf21-infected cells were pelleted at 48 h postinfection and then lysed by sonication on ice in 20 mM Hepes pH 7.5, 250 mM NaCl, and 1 mM DTT with protease inhibitors (Sigma-Aldrich P8340).…”

ACEMBL Tool-Kits for High-Throughput Multigene Delivery and Expression in Prokaryotic and Eukaryotic Hosts

“…Recombinant production offers solutions to these impediments, and a wide range of expression systems are available to produce proteins recombinantly in prokaryotic and eukaryotic hosts [31,33,[34][35][36][37][38][39][40][41][42][43][44][45]. Recombinant expression systems share in common that one or several DNA segments encoding for proteins, protein domains or multicomponent protein complexes are typically combined with DNA elements including DNAs that control transcription (promoters, terminators, others) and translation (ribosome binding sites, ShineDalgarno sequences, Kozak consensus sequences, enhancers, others) and inserted into a functional DNA module (plasmid, cosmid, artificial chromosome, genome, others).…”