biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes - PubMed (original) (raw)
biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes
Florian Weissmann et al. Proc Natl Acad Sci U S A. 2016.
Abstract
Analyses of protein complexes are facilitated by methods that enable the generation of recombinant complexes via coexpression of their subunits from multigene DNA constructs. However, low experimental throughput limits the generation of such constructs in parallel. Here we describe a method that allows up to 25 cDNAs to be assembled into a single baculoviral expression vector in only two steps. This method, called biGBac, uses computationally optimized DNA linker sequences that enable the efficient assembly of linear DNA fragments, using reactions developed by Gibson for the generation of synthetic genomes. The biGBac method uses a flexible and modular "mix and match" approach and enables the generation of baculoviruses from DNA constructs at any assembly stage. Importantly, it is simple, efficient, and fast enough to allow the manual generation of many multigene expression constructs in parallel. We have used this method to generate and characterize recombinant forms of the anaphase-promoting complex/cyclosome, cohesin, and kinetochore complexes.
Keywords: Gibson assembly; baculovirus-insect cell expression; protein complexes.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Schematic representation of the biGBac assembly procedure. (A) A GEC consisting of polyhedrin promoter (polh), cDNA of a GOI, and SV40-terminator (term) is amplified by PCR from pLIB using predefined oligonucleotide sets (Cas_for/rev) to introduce specific linker sequences (Greek letters). (B) First assembly step shown for pBIG1a and five GECs. Linearized vector and PCR products are recombined in a Gibson assembly reaction to a circular product containing a PGC. The positions of SwaI and PmeI restriction sites, which can be used for the analysis of DNA constructs in conjunction with gel electrophoresis, are indicated. (C) Second assembly step shown for pBIG2abcde and five PGCs in pBIG1 vectors. The six plasmids are mixed, digested with PmeI, and recombined in a Gibson assembly reaction. AmpR, ampicillin resistance; CamR, chloramphenicol resistance; SpecR, spectinomycin resistance.
Fig. S1.
Generation of optimized DNA linker sequences. To generate optimized homology sequences for a six-fragment Gibson assembly reaction, 30,000 random DNA sequences of 15 nt in length and defined base composition were generated, and the sequences AAAC and GTTT required for compatibility with PmeI digestion were added to their 5′ and 3′ ends, respectively. Melting temperatures were predicted, and only sequences within the melting temperature range of 59–63 °C were included in further analyses (18,668 sequences). Sequences with a high probability of secondary structure formation on either strand were excluded using RNAfold (11,864 sequences left). The sequence set was further analyzed for secondary structure formation with Zipfold using parameters that fit with Gibson reaction conditions. Only sequences for which both strands were very unlikely to form secondary structures in Gibson assembly reactions [minimum free energy (MFE) ≥ +1.5 kcal/mol] remained in the sequence set (325 sequences). To pick a set of sequences that provides highest specificity in Gibson reactions (i.e., no false annealing), hybrid structures for each combination of two sequences out of the set of 325 sequences and their reverse complements were predicted using RNAcofold. Free energy values of the MFE structure of each hybrid were entered into a 650 × 650 MFE matrix. An algorithm was written in python to build sequence sets in which no pairwise interactions violate an MFE threshold. When setting the threshold to MFE ≥ –3.6 kcal/mol, it was possible to pick exactly one set of six sequences. This set is used as linker sequences in the second assembly step (linkers A, B, C, D, E, and F). This set was manually slightly modified to generate a set that is applicable in the first assembly step (linearization with SwaI), while maintaining all thermodynamic criteria (linkers α, β, γ, δ, ε, and ω).
Fig. S2.
pLIB vector. (A) Schematic representation of pLIB. The cDNA of a GOI is inserted between the polyhedrin promoter (polh) and the SV40 terminator sequence (term). pLIB can be maintained with Ampicillin (AmpR resistance gene). All biGBac vectors (pLIB, pBIG1, pBIG2) contain Tn7 elements (Tn7L, Tn7R) and a gentamicin resistance gene (GentaR) for generation of baculoviruses and a LoxP site for compatibility with Multibac donor plasmids. (B) DNA sequence of empty pLIB from LoxP site to Tn7L element. For generation of library constructs using Gibson reactions, a stock of linearized pLIB can be generated by BamHI/HindIII digestion and a cDNA can be inserted via a Gibson reaction (see Materials and Methods for good homology sequences). Alternatively the MCS can be used to insert a cDNA by conventional restriction/ligation cloning. It is recommended to not clone ORFs in-frame with the mutated polyhedrin start codon (mut. polh start) to avoid the possibility of leaky expression that might lead to N-terminal extensions. The binding sites of the predefined oligonucleotide set (Table S1) for amplification of GECs are indicated (Cas_for/Cas_rev).
Fig. S3.
pBIG1 vectors. (A) Schematic representation of pBIG1 vectors. pBIG1 vectors can be maintained with Spectinomycin (SpecR resistance gene) or Ampicillin (AmpR resistance gene). For the selection in the first assembly step, Spectinomycin is used. Stocks of linearized pBIG1 cloning vectors are generated by SwaI digestion. This results in linear vector backbone with linker sequences α and ω at the fragment ends. After the first assembly step, the generated PGCs can be released from the vector backbone by PmeI digestion. The five pBIG1 vectors differ only in the linker sequences (A, B, C, D, E, and F) next to the PmeI sites as indicated. All biGBac vectors (pLIB, pBIG1, pBIG2) contain Tn7 elements (Tn7L, Tn7R) and a Gentamicin resistance gene (GentaR) for generation of baculoviruses and a LoxP site for compatibility with Multibac donor plasmids. (B) DNA sequence of pBIG1a shown from the LoxP site to Tn7L element. The positions of α, ω and A, B linker sequences as well as of the restriction sites SwaI, PmeI, and PacI are shown. The linker sequences of the first and the second assembly step are separated by spacer sequences (derived from HSVtk terminator sequence in pFL) to avoid interference of the two linker sequence sets in Gibson reactions.
Fig. S4.
pBIG2 vectors. (A) Schematic representation of pBIG2 vectors. pBIG2 vectors are maintained with Chloramphenicol (CamR resistance gene) or Ampicillin (AmpR resistance gene). For the selection in the second assembly step, Chloramphenicol is used. Stocks of linearized pBIG2 cloning vectors are generated by PmeI digestion. This results in linear vector backbone with linker sequences on both ends. The four pBIG2 vectors contain linker sequence A on one end and differ only in the linker sequence on the other side (C, D, E, or F as indicated). All biGBac vectors (pLIB, pBIG1, pBIG2) contain Tn7 elements (Tn7L, Tn7R) and a Gentamicin resistance gene (GentaR) for generation of baculoviruses and a LoxP site for compatibility with Multibac donor plasmids. (B) DNA sequence of pBIG2ab shown from the LoxP site to Tn7L element. The positions of linker sequences A and C and the restriction sites PmeI and PacI are shown.
Fig. 2.
Analysis of biGBac constructs. (A) Analysis of DNA constructs for the expression of APC/C–CDH1–EMI1–SKP1 by restriction digest and gel electrophoresis. The 17 GECs were distributed over the five pBIG1 vectors a–e (FW_I-1/2/3/4/5; Table S3). Correct constructs were analyzed by PmeI or SwaI digestion, releasing PGCs or GECs from the vector “backbone,” respectively, followed by gel electrophoresis and ethidium bromide staining. The five PGCs were recombined with a pBIG2abcde vector (FW_II-1), and a correctly assembled construct is shown before (–) and after digestion with PacI or SwaI, releasing PGCs or GECs, respectively. (B) SwaI digestion of 13 pBIG2 constructs coding for APC/C, subcomplexes, or mutant complexes (FW_II-2–FW_II-14; Table S4). *SwaI digestion of pBIG2 constructs generates additional fragments of 0.3 kb containing spacer sequences between PGCs.
Fig. 3.
Functional and structural characterization of protein complexes expressed from biGBac constructs. (A) Coomassie-stained 4–12% gradient SDS/PAGE gel of purified APC/C alone and bound to CDH1/EMI1/SKP1 (“APC/C-EMI1”). (B) Coomassie-stained 8% SDS/PAGE gel of samples shown in A. *The Strep-tag on APC4 is cleaved in the APC/C sample but not in the APC/C-EMI1 sample. (C) Ubiquitination assay with fluorescein-labeled Cyclin BNTD (CycBNTD*) as substrate. APC/C, coactivator CDH1, E2 ubiquitin-conjugating enzymes UBE2C/UBE2S, and inhibitor EMI1–SKP1 were added as indicated. (D) Single-particle reconstruction by cryo-EM of recombinant APC/C–CDH1–EMI1–SKP1 (“APC/C–EMI1”). (E) HeLa cell APC/C bound to recombinant CDH1 and EMI1-SKP1 (“APC/C-EMI1”) negative stain EM structure data from ref. for comparison. (F) SwaI digestion of pBIG1 constructs coding for wild-type cohesin tetramer (wt; FW_I-16), Walker A double mutant (KA; FW_I-17), and Walker B double mutant (EQ; FW_I-18). (G) SDS/PAGE and silver staining of purified cohesin complexes. (H and I) ATPase activity of cohesin. Thin-layer chromatography (I) and quantification (H) of [γ-32P]-ATP hydrolysis. (J) Representative micrographs of wild-type cohesin tetramers after rotary-shadowing EM. (K) SwaI digestion of yeast kinetochore constructs N (FW_I-19), M (FW_I-20), MN (FW_II-15), and KMN (FW_II-16). †Dsn1 contains a His-tag in M (FW_I-20) but not in MN (FW_II-15) and KMN (FW_II-16). (L) Coomassie-stained SDS/PAGE gel of purified yeast kinetochore subcomplexes. ‡Marked bands were found in samples containing Ndc80-His but not in its absence, suggesting that these represent degradation products of Ndc80-His. (M) Coomassie-stained SDS/PAGE gel of SEC fractions of the KMN complex using a Superose 6 10/300GL column.
Fig. S5.
Examples for construct analysis. (A) Analytical digest of a pBIG1 construct. Six clones of FW_I-23 (pBIG1b:Dsn1/Mtw1/Nnf1/Nsl1/Spc105-Flag) were digested by SwaI or PmeI and analyzed on a 0.8% agarose gel. The expected sizes of pBIG1 vector backbone and GECs after SwaI digestion and of pBIG1 vector backbone and the PGC after PmeI digestion are indicated. Note that spacer sequences (see Fig. S3) stay with the vector backbone after SwaI digestion (5.9 kb) and with the PGC after PmeI digestion (backbone 5.5 kb). Clones 2 and 5 show the correct restriction pattern. (B) Analytical SwaI digest of pBIG2 constructs. Six clones of FW_II-15 (Kinetochore MN) and six clones of FW_II-16 (Kinetochore KMN) were digested with SwaI and analyzed on a 1% agarose gel. The expected sizes of pBIG2 vector backbone and GECs are indicated. Note that SwaI digestion of pBIG2 constructs yields an additional fragment of 0.3 kb consisting mainly of spacer sequences between PGCs. All clones show the correct restriction pattern. (C) Analytical PacI digest of a pBIG2 construct. Six clones of FW_II-1 (APC/C-Emi1) were digested with PacI and analyzed on a 0.4% agarose gel. The expected sizes of pBIG2 vector backbone and PGCs are indicated. All clones show the correct restriction pattern.
References
- Alberts B. The cell as a collection of protein machines: Preparing the next generation of molecular biologists. Cell. 1998;92(3):291–294. -PubMed
- Berger I, Fitzgerald DJ, Richmond TJ. Baculovirus expression system for heterologous multiprotein complexes. Nat Biotechnol. 2004;22(12):1583–1587. -PubMed
- Fitzgerald DJ, et al. Protein complex expression by using multigene baculoviral vectors. Nat Methods. 2006;3(12):1021–1032. -PubMed
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