ArticleMethods in enzymology2017
MacroBac: New Technologies for Robust and Efficient Large-Scale Production of Recombinant Multiprotein Complexes.
Article in Methods in enzymology, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 91 papers.
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Who cites it
91 citing papers in PubMed.
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- Structural basis for fork reversal and RAD51 regulation by the SCF ubiquitin ligase complex of F-box helicase 1.Nature communications · 2026Article
- Molecular basis of XPF-ERCC1 targeting to SLX4-dependent DNA repair pathways.Nature communications · 2025Article
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- Structure of a transcribing Pol II-DSIF-SPT6-U1 snRNP complex.Nature communications · 2025Article
- Distinct roles of the two BRCA2 DNA-binding domains in DNA damage repair and replication fork preservation.Cell reports · 2025Article
- Structural basis of human CHD1 nucleosome recruitment and pausing.Molecular cell · 2025Article
- Molecular insights into the overall architecture of human rixosome.Nature communications · 2025Article
- Molecular basis for the interaction between Saccharomyces cerevisiae Rtt103 and the Rat1-Rai1 complex.Nature communications · 2025Article
31 more citing papers are in PubMed but not listed here.
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6 authors.
Funding
Abstract
Recombinant expression of large, multiprotein complexes is essential and often rate limiting for determining structural, biophysical, and biochemical properties of DNA repair, replication, transcription, and other key cellular processes. Baculovirus-infected insect cell expression systems are especially well suited for producing large, human proteins recombinantly, and multigene baculovirus systems have facilitated studies of multiprotein complexes. In this chapter, we describe a multigene baculovirus system called MacroBac that uses a Biobricks-type assembly method based on restriction and ligation (Series 11) or ligation-independent cloning (Series 438). MacroBac cloning and assembly is efficient and equally well suited for either single subcloning reactions or high-throughput cloning using 96-well plates and liquid handling robotics. MacroBac vectors are polypromoter with each gene flanked by a strong polyhedrin promoter and an SV40 poly(A) termination signal that minimize gene order expression level effects seen in many polycistronic assemblies. Large assemblies are robustly achievable, and we have successfully assembled as many as 10 genes into a single MacroBac vector. Importantly, we have observed significant increases in expression levels and quality of large, multiprotein complexes using a single, multigene, polypromoter virus rather than coinfection with multiple, single-gene viruses. Given the importance of characterizing functional complexes, we believe that MacroBac provides a critical enabling technology that may change the way that structural, biophysical, and biochemical research is done.
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