MultiBac: expanding the research toolbox for multiprotein complexes
2011; Elsevier BV; Volume: 37; Issue: 2 Linguagem: Inglês
10.1016/j.tibs.2011.10.005
ISSN1362-4326
AutoresChristoph Bieniossek, Tsuyoshi Imasaki, Yuichiro Takagi, Imre Berger,
Tópico(s)Virus-based gene therapy research
ResumoProtein complexes composed of many subunits carry out most essential processes in cells and, therefore, have become the focus of intense research. However, deciphering the structure and function of these multiprotein assemblies imposes the challenging task of producing them in sufficient quality and quantity. To overcome this bottleneck, powerful recombinant expression technologies are being developed. In this review, we describe the use of one of these technologies, MultiBac, a baculovirus expression vector system that is particularly tailored for the production of eukaryotic multiprotein complexes. Among other applications, MultiBac has been used to produce many important proteins and their complexes for their structural characterization, revealing fundamental cellular mechanisms. Protein complexes composed of many subunits carry out most essential processes in cells and, therefore, have become the focus of intense research. However, deciphering the structure and function of these multiprotein assemblies imposes the challenging task of producing them in sufficient quality and quantity. To overcome this bottleneck, powerful recombinant expression technologies are being developed. In this review, we describe the use of one of these technologies, MultiBac, a baculovirus expression vector system that is particularly tailored for the production of eukaryotic multiprotein complexes. Among other applications, MultiBac has been used to produce many important proteins and their complexes for their structural characterization, revealing fundamental cellular mechanisms. Our understanding of cellular function has remarkably expanded in recent years, brought about by technological advances in DNA and protein analysis [1Nie Y. et al.Getting a grip on complexes.Curr. Genomics. 2009; 10: 558-572Crossref PubMed Scopus (23) Google Scholar]. The sequencing of complete genomes, such as the human genome, has set the stage to address proteome-wide interaction studies, which have revealed that proteins do not typically exist as isolated entities [2Puig O. et al.The tandem affinity purification (TAP) method: a general procedure of protein complex purification.Methods. 2001; 24: 218-229Crossref PubMed Scopus (1446) Google Scholar, 3Gavin A.C. et al.Functional organization of the yeast proteome by systematic analysis of protein complexes.Nature. 2002; 415: 141-147Crossref PubMed Scopus (4063) Google Scholar, 4Gavin A.C. et al.Proteome survey reveals modularity of the yeast cell machinery.Nature. 2006; 440: 631-636Crossref PubMed Scopus (2165) Google Scholar]. Rather, they are assembled in complex molecular machines consisting of numerous proteins and, often, other biomolecules (such as nucleic acids, sugars, lipids and small molecules), arranged into functional modules that catalyze essential cellular processes. This molecular organization has been recently termed 'protein sociology' [5Robinson C.V. et al.The molecular sociology of the cell.Nature. 2007; 450: 973-982Crossref PubMed Scopus (435) Google Scholar]. Understanding cellular processes requires detailed knowledge of the 3D structure of the molecules involved, and the parameters and architectural features that dictate their interaction. Structural genomics efforts strive to analyze comprehensively single proteins and protein domain structures on a whole-genome scale, and have provided atomic structures of thousands of protein structures and folds. Furthermore, the architectures of essential macromolecular complexes, such as ribosomes, nucleosomes and RNA polymerases, have been revealed at near atomic resolution, providing a wealth of structural detail and crucial insight into the functions of these multicomponent machines [6Korostelev A. Noller H.F. The ribosome in focus: new structures bring new insights.Trends Biochem. 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Notwithstanding, our molecular understanding of the very large number of protein complexes in the cell remains limited to a handful of examples for which detailed near-atomic structures are known. This is mostly because of the difficulty in obtaining samples in sufficient quality and quantity for molecular studies. Many essential complexes remain intractable because they exist in very low amounts in their endogenous host, which hinders their purification from native source material. Recombinant overproduction can resolve this bottleneck, and numerous expression systems, mostly for heterologous protein expression in Escherichia coli, have been developed and refined over the past few decades. More recently, E. coli expression systems have been designed for coexpression of several proteins by using polycistronic mRNA transcripts, or two or more plasmids that coexist in the same cell [10Busso D. et al.Expression of protein complexes using multiple Escherichia coli protein co-expression systems: A benchmarking study.J. Struct. Biol. 2011; 175: 159-170Crossref PubMed Scopus (33) Google Scholar, 11Diebold M.N. et al.Deciphering correct strategies for multiprotein complex assembly by co-expression: application to complexes as large as the histone octamer.J. Struct. Biol. 2011; 175: 178-188Crossref PubMed Scopus (96) Google Scholar] (Box 1). However, many eukaryotic protein complexes cannot be produced efficiently in E. coli. They may contain subunits that are too large for the E. coli transcription and translation machinery, or may require either specific chaperone systems for proper folding or protein modifications (such as phosphorylation or acetylation) that E. coli cannot provide. Thus, the successful overproduction of these complexes, which is required to decipher their structure and function, depends on the availability of powerful eukaryotic expression technologies. In this review, we describe MultiBac, a recent eukaryotic expression system that is specifically designed to tackle and overcome this crucial production bottleneck [12Berger I. et al.Baculovirus expression system for heterologous multiprotein complexes.Nat. Biotechnol. 2004; 22: 1583-1587Crossref PubMed Scopus (357) Google Scholar, 13Fitzgerald D.J. et al.Protein complex expression by using multigene baculoviral vectors.Nat. Methods. 2006; 3: 1021-1032Crossref PubMed Scopus (281) Google Scholar]. We summarize the technology concepts underlying MultiBac and review its wide range of applications.Box 1Coexpression toolbox for production of protein complexesExpression systems for production of protein complexes in E. coli frequently make use of polycistronic expression cassettes with several genes of interest, spaced apart by ribosome binding sites (Shine–Dalgarno sequences), placed under the control of a single promoter, and typically followed by a sequence for efficient termination of mRNA transcription [10Busso D. et al.Expression of protein complexes using multiple Escherichia coli protein co-expression systems: A benchmarking study.J. Struct. Biol. 2011; 175: 159-170Crossref PubMed Scopus (33) Google Scholar, 11Diebold M.N. et al.Deciphering correct strategies for multiprotein complex assembly by co-expression: application to complexes as large as the histone octamer.J. Struct. Biol. 2011; 175: 178-188Crossref PubMed Scopus (96) Google Scholar, 21Bieniossek C. et al.Automated unrestricted multigene recombineering for multiprotein complex production.Nat. Methods. 2009; 6: 447-450Crossref PubMed Scopus (82) Google Scholar, 60Tolia N.H. Joshua-Tor L. Strategies for protein coexpression in Escherichia coli.Nat. Methods. 2006; 3: 55-64Crossref PubMed Scopus (197) Google Scholar, 61Tan S. et al.The pST44 polycistronic expression system for producing protein complexes in Escherichia coli.Protein Expr. Purif. 2005; 40: 385-395Crossref PubMed Scopus (137) Google Scholar, 62Romier C. et al.Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies.Acta Crystallogr. D: Biol. Crystallogr. 2006; 62: 1232-1242Crossref PubMed Scopus (109) Google Scholar] (Figure Ia). In eukaryotic hosts, an analogous design can involve internal ribosomal entry sites (IRESs), which are inserted between gene coding regions under control of a single promoter [24Kieft J.S. Viral IRES RNA structures and ribosome interactions.Trends Biochem. Sci. 2008; 33: 274-283Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 25Komar A.A. Hatzoglou M. Cellular IRES-mediated translation.Cell Cycle. 2011; 10: 229-240Crossref PubMed Scopus (303) Google Scholar, 26Semler B.L. Waterman M.L. IRES mediated pathways to polysomes: nuclear versus cytoplasmic routes.Trends Microbiol. 2008; 16: 1-5Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 27Chen W.S. et al.Development of a prokaryotic-like polycistronic baculovirus expression vector by the linkage of two internal ribosome entry sites.J. Virol. Methods. 2009; 159: 152-159Crossref PubMed Scopus (10) Google Scholar] (Figure Ib). IRESs exist in the 5′-untranslated regions of many viral and cellular mRNAs [24Kieft J.S. Viral IRES RNA structures and ribosome interactions.Trends Biochem. Sci. 2008; 33: 274-283Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar], and facilitate cap-independent translation by recruiting ribosomes for efficient protein production. IRES sequences differ greatly and exhibit species specificity [26Semler B.L. Waterman M.L. IRES mediated pathways to polysomes: nuclear versus cytoplasmic routes.Trends Microbiol. 2008; 16: 1-5Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar]. For example, IRES elements from encephalomyocarditis virus (EMCV) work well in mammalian cells, whereas IRESs from Perina nuda virus (PhV) and Rhopalosiphum padi virus (RhPV) have been successfully used for protein complex production in insect cells [27Chen W.S. et al.Development of a prokaryotic-like polycistronic baculovirus expression vector by the linkage of two internal ribosome entry sites.J. Virol. Methods. 2009; 159: 152-159Crossref PubMed Scopus (10) Google Scholar].Polyproteins are long polypeptides containing individual proteins spaced by specific proteolytic cleavage sites. Certain viruses, such as coronavirus, produce their entire proteome by proteolytic processing of polyproteins encoded by a few open reading frames. Polyprotein approaches have proven to be particularly powerful for balancing the stoichiometry of coexpressed proteins [22Vijayachandran L.S. et al.Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells.J. Struct. Biol. 2011; 175: 198-208Crossref PubMed Scopus (75) Google Scholar, 28Szymczak A.L. et al.Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.Nat. Biotechnol. 2004; 22: 589-594Crossref PubMed Scopus (947) Google Scholar, 29Kim J.H. et al.High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.PLoS ONE. 2011; 6: e18556Crossref PubMed Scopus (969) Google Scholar, 30Chen X. et al.TEV protease-facilitated stoichiometric delivery of multiple genes using a single expression vector.Protein Sci. 2010; 19: 2379-2388Crossref PubMed Scopus (27) Google Scholar] (Figure Ic). Polyprotein constructions can involve self-cleaving peptides found in picornavirus (called P2A peptides) or variants thereof [28Szymczak A.L. et al.Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.Nat. Biotechnol. 2004; 22: 589-594Crossref PubMed Scopus (947) Google Scholar, 29Kim J.H. et al.High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.PLoS ONE. 2011; 6: e18556Crossref PubMed Scopus (969) Google Scholar]. Alternatively, constructions can be used that mimic open reading frames in positive-sense single-stranded RNA viruses and provide a highly specific protease gene together with genes of interest arranged in a single large open reading frame [22Vijayachandran L.S. et al.Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells.J. Struct. Biol. 2011; 175: 198-208Crossref PubMed Scopus (75) Google Scholar, 30Chen X. et al.TEV protease-facilitated stoichiometric delivery of multiple genes using a single expression vector.Protein Sci. 2010; 19: 2379-2388Crossref PubMed Scopus (27) Google Scholar]. Individual proteins are then liberated from the encoded polyprotein by the protease, which cleaves the proteolytic sites placed between the protein subunits (Figure Ic).The elucidation of protein–protein interactions in complexes is of crucial importance for many applications including structural biology. A novel approach, CoESPRIT, utilizes library-based construct screening for the identification and expression of soluble protein complexes in E. coli [31An Y. et al.CoESPRIT: a library-based construct screening method for identification and expression of soluble protein complexes.PLoS ONE. 2011; 6: e16261Crossref PubMed Scopus (15) Google Scholar]. In CoESPRIT, a subunit of a (putative) protein complex is provided as bait. The interacting partner is provided in the form of a random incremental library generated by exonuclease digestion of the full-length gene. Prior input from bioinformatics analyses such as homology alignments or domain identification is not required for this approach. Coexpression of the library with the bait protein allows identification of soluble complexes by immunofluorescence-assisted colony screening using labeled antibody markers against affinity tags present on the proteins screened. Production of protein complex from high-expressor colonies thus identified can then be scaled up to milligram amounts for high-resolution studies by NMR or X-ray crystallography [31An Y. et al.CoESPRIT: a library-based construct screening method for identification and expression of soluble protein complexes.PLoS ONE. 2011; 6: e16261Crossref PubMed Scopus (15) Google Scholar]. Expression systems for production of protein complexes in E. coli frequently make use of polycistronic expression cassettes with several genes of interest, spaced apart by ribosome binding sites (Shine–Dalgarno sequences), placed under the control of a single promoter, and typically followed by a sequence for efficient termination of mRNA transcription [10Busso D. et al.Expression of protein complexes using multiple Escherichia coli protein co-expression systems: A benchmarking study.J. Struct. Biol. 2011; 175: 159-170Crossref PubMed Scopus (33) Google Scholar, 11Diebold M.N. et al.Deciphering correct strategies for multiprotein complex assembly by co-expression: application to complexes as large as the histone octamer.J. Struct. Biol. 2011; 175: 178-188Crossref PubMed Scopus (96) Google Scholar, 21Bieniossek C. et al.Automated unrestricted multigene recombineering for multiprotein complex production.Nat. Methods. 2009; 6: 447-450Crossref PubMed Scopus (82) Google Scholar, 60Tolia N.H. Joshua-Tor L. Strategies for protein coexpression in Escherichia coli.Nat. Methods. 2006; 3: 55-64Crossref PubMed Scopus (197) Google Scholar, 61Tan S. et al.The pST44 polycistronic expression system for producing protein complexes in Escherichia coli.Protein Expr. Purif. 2005; 40: 385-395Crossref PubMed Scopus (137) Google Scholar, 62Romier C. et al.Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies.Acta Crystallogr. D: Biol. Crystallogr. 2006; 62: 1232-1242Crossref PubMed Scopus (109) Google Scholar] (Figure Ia). In eukaryotic hosts, an analogous design can involve internal ribosomal entry sites (IRESs), which are inserted between gene coding regions under control of a single promoter [24Kieft J.S. Viral IRES RNA structures and ribosome interactions.Trends Biochem. Sci. 2008; 33: 274-283Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 25Komar A.A. Hatzoglou M. Cellular IRES-mediated translation.Cell Cycle. 2011; 10: 229-240Crossref PubMed Scopus (303) Google Scholar, 26Semler B.L. Waterman M.L. IRES mediated pathways to polysomes: nuclear versus cytoplasmic routes.Trends Microbiol. 2008; 16: 1-5Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 27Chen W.S. et al.Development of a prokaryotic-like polycistronic baculovirus expression vector by the linkage of two internal ribosome entry sites.J. Virol. Methods. 2009; 159: 152-159Crossref PubMed Scopus (10) Google Scholar] (Figure Ib). IRESs exist in the 5′-untranslated regions of many viral and cellular mRNAs [24Kieft J.S. Viral IRES RNA structures and ribosome interactions.Trends Biochem. Sci. 2008; 33: 274-283Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar], and facilitate cap-independent translation by recruiting ribosomes for efficient protein production. IRES sequences differ greatly and exhibit species specificity [26Semler B.L. Waterman M.L. IRES mediated pathways to polysomes: nuclear versus cytoplasmic routes.Trends Microbiol. 2008; 16: 1-5Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar]. For example, IRES elements from encephalomyocarditis virus (EMCV) work well in mammalian cells, whereas IRESs from Perina nuda virus (PhV) and Rhopalosiphum padi virus (RhPV) have been successfully used for protein complex production in insect cells [27Chen W.S. et al.Development of a prokaryotic-like polycistronic baculovirus expression vector by the linkage of two internal ribosome entry sites.J. Virol. Methods. 2009; 159: 152-159Crossref PubMed Scopus (10) Google Scholar]. Polyproteins are long polypeptides containing individual proteins spaced by specific proteolytic cleavage sites. Certain viruses, such as coronavirus, produce their entire proteome by proteolytic processing of polyproteins encoded by a few open reading frames. Polyprotein approaches have proven to be particularly powerful for balancing the stoichiometry of coexpressed proteins [22Vijayachandran L.S. et al.Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells.J. Struct. Biol. 2011; 175: 198-208Crossref PubMed Scopus (75) Google Scholar, 28Szymczak A.L. et al.Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.Nat. Biotechnol. 2004; 22: 589-594Crossref PubMed Scopus (947) Google Scholar, 29Kim J.H. et al.High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.PLoS ONE. 2011; 6: e18556Crossref PubMed Scopus (969) Google Scholar, 30Chen X. et al.TEV protease-facilitated stoichiometric delivery of multiple genes using a single expression vector.Protein Sci. 2010; 19: 2379-2388Crossref PubMed Scopus (27) Google Scholar] (Figure Ic). Polyprotein constructions can involve self-cleaving peptides found in picornavirus (called P2A peptides) or variants thereof [28Szymczak A.L. et al.Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.Nat. Biotechnol. 2004; 22: 589-594Crossref PubMed Scopus (947) Google Scholar, 29Kim J.H. et al.High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.PLoS ONE. 2011; 6: e18556Crossref PubMed Scopus (969) Google Scholar]. Alternatively, constructions can be used that mimic open reading frames in positive-sense single-stranded RNA viruses and provide a highly specific protease gene together with genes of interest arranged in a single large open reading frame [22Vijayachandran L.S. et al.Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells.J. Struct. Biol. 2011; 175: 198-208Crossref PubMed Scopus (75) Google Scholar, 30Chen X. et al.TEV protease-facilitated stoichiometric delivery of multiple genes using a single expression vector.Protein Sci. 2010; 19: 2379-2388Crossref PubMed Scopus (27) Google Scholar]. Individual proteins are then liberated from the encoded polyprotein by the protease, which cleaves the proteolytic sites placed between the protein subunits (Figure Ic). The elucidation of protein–protein interactions in complexes is of crucial importance for many applications including structural biology. A novel approach, CoESPRIT, utilizes library-based construct screening for the identification and expression of soluble protein complexes in E. coli [31An Y. et al.CoESPRIT: a library-based construct screening method for identification and expression of soluble protein complexes.PLoS ONE. 2011; 6: e16261Crossref PubMed Scopus (15) Google Scholar]. In CoESPRIT, a subunit of a (putative) protein complex is provided as bait. The interacting partner is provided in the form of a random incremental library generated by exonuclease digestion of the full-length gene. Prior input from bioinformatics analyses such as homology alignments or domain identification is not required for this approach. Coexpression of the library with the bait protein allows identification of soluble complexes by immunofluorescence-assisted colony screening using labeled antibody markers against affinity tags present on the proteins screened. Production of protein complex from high-expressor colonies thus identified can then be scaled up to milligram amounts for high-resolution studies by NMR or X-ray crystallography [31An Y. et al.CoESPRIT: a library-based construct screening method for identification and expression of soluble protein complexes.PLoS ONE. 2011; 6: e16261Crossref PubMed Scopus (15) Google Scholar]. Yeast, mammalian cells and insect cells have been successfully used for recombinant production of eukaryotic proteins [14Nettleship J.E. et al.Recent advances in the production of proteins in insect and mammalian cells for structural biology.J. Struct. Biol. 2010; 172: 55-65Crossref PubMed Scopus (80) Google Scholar]. In particular, the use of insect cell cultures infected by a recombinant baculovirus, carrying the eukaryotic gene of interest, was first demonstrated many years ago [15Smith G.E. et al.Production of human beta interferon in insect cells infected with a baculovirus expression vector.Mol. Cell. Biol. 1983; 3: 2156-2165Crossref PubMed Scopus (739) Google Scholar]. The exciting evolution of baculovirus from a pest control agent to a powerful recombinant protein production tool has recently been reviewed [16Summers M.D. Milestones leading to the genetic engineering of baculoviruses as expression vector systems and viral pesticides.Adv. Virus Res. 2006; 68: 3-73Crossref PubMed Scopus (139) Google Scholar], and baculovirus expression systems have become increasingly popular for many applications [17Kost T.A. et al.Baculovirus as versatile vectors for protein expression in insect and mammalian cells.Nat. Biotechnol. 2005; 23: 567-575Crossref PubMed Scopus (796) Google Scholar, 18Kost T.A. et al.Baculovirus gene delivery: a flexible assay development tool.Curr. Gene Ther. 2010; 10: 168-173Crossref PubMed Scopus (61) Google Scholar, 19Trowitsch S. et al.New baculovirus expression tools for recombinant protein complex production.J. Struct. Biol. 2010; 172: 45-54Crossref PubMed Scopus (147) Google Scholar]. MultiBac is a baculovirus expression system particularly designed for the production of eukaryotic multiprotein complexes with many subunits (Figure 1). It consists of an array of small synthetic DNA plasmids, an engineered baculovirus genome derived from the Autographa californica nuclear polyhedrosis virus (AcNPV; see Glossary) that is used to infect cells of the caterpillar Spodoptera frugiperda, and a set of protocols detailing every step from gene insertion into the plasmids to production of protein complexes in cultured insect cells [19Trowitsch S. et al.New baculovirus expression tools for recombinant protein complex production.J. Struct. Biol. 2010; 172: 45-54Crossref PubMed Scopus (147) Google Scholar, 20Bieniossek C. et al.MultiBac: multigene baculovirus-based eukaryotic protein complex production.Curr. Protoc. Protein Sci. 2008; (Chapter 5, Unit 5.20)Crossref PubMed Scopus (118) Google Scholar]. The presence of many subunits in a protein complex requires the assembly of many encoding genes and their integration into the baculovirus in a multicomponent co-production experiment. This process is laborious and technically challenging using conventional methods that typically involve serial insertion of genes into increasingly large and difficult-to-handle DNA plasmids. MultiBac applies a different concept for multigene assembly that relies on recombination of small, custom-designed, synthetic DNA plasmid molecules (< 3 kb) that are called 'acceptors' and 'donors' (Figure 1a). Acceptors and donors can be easily loaded with one or several genes each, and recombined in a single-step reaction into a multigene construct. Acceptors contain an origin of replication that allows propagation in standard cloning strains of E. coli, whereas donors harbor a conditional origin of replication (derived from phage R6Kγ) that requires the presence of a specific protein (known as π) for replication. This protein is expressed from the pir gene inserted into the chromosome of specifically tailored E. coli strains that are used to propagate donor plasmids [13Fitzgerald D.J. et al.Protein complex expression by using multigene baculoviral vectors.Nat. Methods. 2006; 3: 1021-1032Crossref PubMed Scopus (281) Google Scholar, 20Bieniossek C. et al.MultiBac: multigene baculovirus-based eukaryotic protein complex production.Curr. Protoc. Protein Sci. 2008; (Chapter 5, Unit 5.20)Crossref PubMed Scopus (118) Google Scholar]. Donors and acceptors contain a resistance marker, a short imperfect inverted repeat (LoxP), an expression cassette consisting of a baculoviral promoter (p10 or polh), a DNA segment for inserting heterologous genes, and an efficient signal for eukaryotic polyadenylation (Figure 1a). The expression cassettes are flanked by a homing endonuclease site and a compatible BstXI site, which allow for iterative assembly of several expression cassettes on each plasmid [21Bieniossek C. et al.Automated unrestricted multigene recombineering for multiprotein complex production.Nat. Methods. 2009; 6: 447-450Crossref PubMed Scopus (82) Google Scholar]. Importantly, donors can survive in a pir-negative background only if they are conjoined with an acceptor that provides a nonconditional origin of replication, and this is the central feature that enables flexible and efficient generation of multigene constructs. These are achieved in vitro by Cre recombinase, which fuses one or several donors (each with one or several inserted genes) to a single acceptor in a one-step reaction by conjoining via the LoxP sites; this results in plasmid dimers, trimers and tetramers. The resulting multigene expression constructs are characterized by the precise combinations of resistance markers present on the fusions, and this can be exploited for combinatorial assembly strategies based on multiple antibiotic selection [21Bieniossek C. et al.Automated unrestricted multigene recombineering for multiprotein complex production.Nat. Methods. 2009; 6: 447-450Crossref PubMed Scopus (82) Google Scholar]. The process of inserting genes into acceptors and donors, which can be optionally done by ligation independent methods followed by Cre–LoxP fusion, is termed 'tandem recombineering' [22Vijayachandran L.S. et al.Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells.J. Struct. Biol. 2011; 175: 198-208Crossref PubMed Scopus (75) Google Scholar]. Gene insertions into the MultiBac genome take place in bacterial strains that contain the MultiBac viral genome as an artificial chromosome together with a plasmid encoding the Tn7 transposon enzyme complex. Multigene acceptor–donor fusion constructs are transformed into these bacterial cells, and the Tn7 transposon enzyme inserts the collection of expression cassettes present on the acceptor–donor fusion in a single-step reaction into the Tn7 attachment site engineered into the baculoviral genome (Figure 1b). Productive transposition disrupts a LacZα-encoding gene, which enables blue/white screening of colonies. The MultiBac baculoviral genome has been engineered for improved protein complex production by removing genes encoding viral protease and apoptotic activities, thereby reducing proteolytic breakdown of the heterologous gene products and delaying lysis of the infected cells [12Berger I. et al.Baculovirus expression system for heterologous multiprotein complexes.Nat. Biotechnol. 2004; 22: 1583-1587Crossref PubMed Scopus (357) Google Scholar, 13Fitzgerald D.J. et al.Protein complex expression by using multigene baculoviral vectors.Nat. Methods. 2006; 3: 1021-1032Crossref PubMed Scopus (281) Google Scholar]. As a second site of entry in addition to the Tn7 attachment site, the MultiBac genome contains also a distal LoxP site for adding further functionalities. For example, a gene encoding λ-phosphatase was inserted into this site to remove phosphates from a coexpres
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