Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.
It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.
It is imperative for a research laboratory to be capable of producing large quantities of homogeneous recombinant proteins for biochemical and biophysical characterizations, especially for X-ray crystallographic studies. There are many well-established heterologous expression systems such as Escherichia coli, yeast, insect cells, mammalian cells, plant cells, etc. Among them, the baculovirus-mediated insect cell expression system is one of the most commonly used techniques to produce large quantities of structurally folded large-sized recombinant eukaryotic proteins for protein crystallization1.
The expression vectors of the baculovirus expression system are engineered to contain a strong polyhedrin or P10 promoter to produce a high yield of recombinant intracellular proteins2,3. To make a recombinant baculovirus, the gene of interest is cloned into an insect vector containing the polyhedrin (polh) locus of the Autographa californica multi-nucleopolyhedroviral genome. The resulting construct is then sequenced and its correct open reading frame (ORF) is verified. The correct construct is then introduced into the host insect cell through the process of transfection. The gene of interest is inserted into the viral genome by homologous recombination. This event results in the production of the recombinant viral genome, which then replicates to produce recombinant budded virus particles1.
The insect cells that are most commonly used in the expression system are Sf9 and High Five (Hi5) cells. Sf9 cells are a clonal isolate of Sf21, derived from the pupal ovarian cells of Spodoptera frugiperda, and Hi5 cells are a clonal isolate derived from the parental Trichoplusia ni ovarian cell line TN-3684,5. Co-transfections, virus amplification, and plaque assays are conducted on Sf9 cells, while Hi5 cells are typically selected to produce higher quantities of recombinant proteins6. It is worth noting that the Hi5 cells are not suitable for the generation and amplification of virus progenies because of their tendency to produce mutant viruses. Traditionally, a temperature range of 25 – 30 °C is considered to be good for the cultivation of insect cells. However, it has been reported that 27 – 28 °C is the optimal temperature for the insect cell growth and infection7,8.
The introduction of a strong signal sequence preceding the gene is needed for the high expression of the secreted proteins. The signal sequence would efficiently guide the translated recombinant protein into the endoplasmic reticulum for protein secretion and post-translational modifications necessary for proper folding and stabilization3. Signal peptide sequences, such as the baculovirus envelop protein GP64/67, honeybee melittin, and others, have been chosen to facilitate the expression of secretory recombinant proteins in the baculovirus-mediated expression systems3. The introduction of the signal peptide of GP67 has been shown to improve the expression yield of a secreted recombinant protein, in comparison to using the intrinsic signal peptide of the target gene9. Hemolin is a hemolymph protein of the giant silk moth Hyalophora cecropia, induced upon bacteria infection10. Due to the relatively high level of induced expression, the signal peptide sequence of the gene can be used to mediate the secretion expression of the recombinant proteins in the baculovirus-insect cell system.
The A. thaliana Tracheary Element Differentiation Inhibitory Factor Receptor (TDR) and Pollen Receptor Kinase 3 (PRK3) both belong to the plant Leucine-rich Repeat Receptor-like Kinase (LRR-RLK) family of proteins11,12. In order to study the structure and function of this family of plant receptor proteins, as well as to facilitate the structural and biochemical characterization of other plant secreted proteins, the baculovirus-insect cell expression system has been modified to improve protein quality and production yield. The extracellular domains of both TDR and PRK3 have successfully been expressed using two modified expression vectors in the baculovirus-insect cell expression system. Both the extracellular domains of TDR and PRK3 proteins have been crystallized. This article reports the expression and purification of large amounts of recombinant secreted plant proteins with two modified baculovirus expression vectors by incorporating either a GP67 or a hemolin signal sequence between the promoter and multiple cloning sites.
NOTE: An insect cell/baculovirus system with modified expression vectors for secretory plant protein expression and crystallization is used.
1. Modification of a Baculovirus Expression Vector with the GP67 Signal Peptide for Plant Protein Secretion Expression
2. Modification of a Baculovirus Expression Vector with the Insect Hemolin Signal Peptide for Plant Protein Secretion Expression
3. Production and Amplification of Baculovirus Constructs Harboring the Recombinant Protein Expression Cassette
4. Protein Expression, Purification with NiNTA, and Crystallization
As shown in Figure 1, two modified pFastBac1 baculovirus expression vectors were used to express the secreted proteins with either the GP67 or the hemolin signal sequence to replace the intrinsic signal sequence of the target gene. The viral GP67 and the insect hemolin genes have been demonstrated to have high secretion expression levels in the cells. Fusion proteins with either of these two signal sequences are expected to have greatly improved secretion expression levels. Identical multiple cloning sites (MCS) were engineered in these two modified vectors. A NotI site was deliberately placed on the most 5'-side of the MCS, because NotI has an eight-nucleotide sequence rather than the most common six-nucleotide sequence present in many other restriction sites. As such, a NotI site is less present in the target genes than most other restriction sites, making restriction digestion and the cloning of most target genes more feasible in comparison to using other restriction sites. Cloning the target gene between the NotI and a downstream restriction site will introduce only three amino acid residues, GGR, preceding the target gene.
The pBac1-GP67 and pBac1-Hem have been successfully utilized to express the extracellular domains of the A. thaliana receptors TDR and PRK3, respectively (Figure 2). After the recombinant proteins were purified by NiNTA using an engineering C-terminal 6-histidine tag, the average protein yield was consistent around 20 mg/L of Hi5 cells. The purity of each protein is close to or higher than 50%, examined with SDS-PAGE and Coomassie staining.
The recombinant proteins of the extracellular domains of TDR and PRK3 were further purified by size exclusion chromatography (Figure 2). The purified protein was concentrated to 5 mg/mL and then subjected to a crystallization screening. The conditions, which yielded preliminary crystals of each protein, were optimized until protein crystals with a size bigger than 20 µm were observed (Figure 3). The crystal structures of both proteins have been reported11,12.
Figure 1: Maps of the modified pFastBac1 vectors. The DNA sequences shown in these panels were inserted downstream of the polyhedrin promoter of the pFastBac1 vector, to possess the signal peptide sequence and the multiple cloning sites (MCS) for target genes. Both vectors contain the same MCS. (A) This panel shows a sequence of the cloning sites downstream of the polyhedrin promoter in the pBac1-GP67 vector. The translated GP67 signal peptide amino acid sequence is colored in red. Each unique restriction site in the MCS is underlined, with the name of the site labeled above. (B) This panel shows a sequence of the cloning sites downstream of the polyhedrin promoter in the pBac1-Hem vector. The translated hemolin signal peptide amino acid sequence is colored in red. Each unique restriction site in the MCS is underlined, with the name of the site labeled above. Please click here to view a larger version of this figure.
Figure 2: Protein expression with the modified pFastBac1 vectors in the baculovirus-insect cell system. (A) The ectodomain of TDR was expressed in Hi5 insect cells, purified by nickel-affinity and size exclusion chromatography (S), and resolved on SDS-PAGE gel. The nickel resin was washed once with a buffer containing 5 mM imidazole (wash 1), and a second time with 20 mM imidazole (wash 2). (B) This is an SDS-PAGE analysis showing the expression and nickel affinity purification (NiNTA), as well as the size exclusion chromatography of the PRK3 ectodomain. Molecular weight (MW) markers with the corresponding sizes are labeled. The red arrows in each panel denote the expressions and the expected sizes of the recombinant TDR and PRK3 ectodomain proteins. The multi-band natures of the expressed proteins are likely due to heterogeneous glycosylation. Please click here to view a larger version of this figure.
Figure 3: Protein crystals of the extracellular domains of Arabidopsis thaliana TDR and PRK3. (A) This panel shows the protein crystals of the extracellular domains of A. thaliana TDR. (B) This panel shows the protein crystals of the extracellular domains of A. thaliana PRK3. A scale bar is shown below each picture. Please click here to view a larger version of this figure.
Given the diversity in size and stability of the thousands of proteins present in the biological systems, it is often empirical for a research laboratory to decide which heterologous expression system has to be chosen for the expression of a specific protein. The E. coli expression system is often the first choice for protein expression due to the short life cycle of the bacteria, low cost of the culture media, and relative ease to scale up19. For the expression of large eukaryotic proteins with sizes more than 60 kDa, however, using the E. coli system often results in insoluble proteins in the inclusion body or aggregation20. For the expression of those difficult proteins, and for other secreted proteins, the baculovirus-insect cell expression system may be more advantageous than the E coli. Especially when expressing secreted eukaryotic proteins, this modified baculovirus-insect cell expression system could be a preferred choice.
Many secreted eukaryotic proteins, including plant secreted proteins, need complex glycosylation, disulfide formation, and other post-translational modifications during protein folding and secretion21. E. coli systems do not have the cellular machinery to process the complex modifications required for many of the eukaryotic proteins22. Yeast has a relatively more advanced glycosylation system than E. coli23. However, for the expression of the secreted proteins in higher eukaryotic species and plants, the baculovirus-insect cell system presents a significant advantage. Since glycosylation affects protein folding and stability24, many of the secreted recombinant proteins expressed in the E. coli and yeast systems have a low yield and tend to aggregate, presumably due to incorrect protein folding. The baculovirus-insect system has been modified to improve protein glycosylation, which makes it an ideal system for the expression of secreted eukaryotic proteins25. In this study, both the GP67 and the hemolin signal peptides have been successfully used to guide the secretion expression of two plant receptor proteins for protein crystallization. With these studies in mind though, the choice of either signal peptide is a critical step, and it needs more systematic comparative studies with a set of target genes from different organisms.
The idea of using both GP67 and hemolin signal peptides to enhance protein secretion expression was driven by the high expression yields of both genes. However, if the protein secretion and posttranslational modification machinery of the expression host are overloaded with the expressed recombinant proteins, the secreted recombinant proteins may not have adequate modifications, especially glycosylation. Both modified vectors have been used to successfully express numerous plant secretory proteins11,12,26, many of which tend to aggregate, which is probably due to the incorrect or inadequate glycosylation. Therefore, for the expression of those difficult proteins, both strong and weak signal peptide sequences have to be tested and the quality of the expressed recombinant proteins needs to be compared. Keep in mind that a weak signal peptide sequence will lower the overall yield of the protein; however, it may give the secretion machinery of the expression host enough capacity to process the protein secretion and modifications.
In addition to the expression of heterologous secreted proteins in insect cells, the mammalian cell and plant cell expression systems have been used successfully in the overexpression of recombinant mammalian and plant proteins, respectively27,28,29. In comparison to the heterologous systems, the near endogenous expression condition of either the mammalian or the plant secreted proteins will have the almost native modification machineries in the host cells to yield well-folded proteins. The caveat of using the near-native expression system is that the expressed recombinant proteins may interfere with the physiological function of the cells, which may potentially have adverse effects on the final expression yield of the proteins.
The authors have nothing to disclose.
This work was supported by the new faculty startup funds from North Carolina State University for Guozhou Xu.
Incubator shaker | VWR | Model Excella E25 | 27 oC |
Incubator | VWR | Model 2005 | 27 oC |
Centrifuge | BECKMAN | Model J-6 | with a swing bucket rotor |
Herculase II Fusion DNA Polymerase | Agilent | 600677-51 | For PCR amplification of DNA |
Thermal Cycler | BIO-RAD | Model C1000 Touch | For PCR amplification of DNA |
Incubator shaker | New Brunswick Scientific | Model I 24 | For growing baceria culture |
Customer DNA synthesis | GENSCRIPT | ||
BamHI (HF) | New England Biolabs | R3136S | Restriction Endonuclease |
BglII | New England Biolabs | R0144S | Restriction Endonuclease |
NotI (HF) | New England Biolabs | R3189S | Restriction Endonuclease |
XhoI | New England Biolabs | R0146S | Restriction Endonuclease |
T4 DNA ligase | New England Biolabs | M0202T | DNA ligation |
QIAquick Gel Extraction Kit | Qiagen | 28704 | DNA purification from Agorase gel |
QIAprep Spin Miniprep Kit | Qiagen | 27104 | Plasmid DNA purification from bacteria culture |
Agarose | Thermo Fisher Scientific | 15510-019 | For DNA gel electropherosis |
MAX Efficiency DH5α competen cell | Invitrogen | 18-258-012 | For transformation of DNA ligation mixture |
Lennox L LB Broth | Research Product International Corp. | L24066-5000.0 | For making bacteria culture |
Ampicillin sodium salt | Thermo Fisher Scientific | 11593-019 | Antibiotics |
Kanamycin Sulfate | Thermo Fisher Scientific | 15160-054 | Antibiotics |
Tetracycline | Thermo Fisher Scientific | 64-75-5 | Antibiotics |
Gentamicin | Thermo Fisher Scientific | 15710-064 | Antibiotics |
MAX Efficiency DH10Bac competent cells | Thermo Fisher Scientific | 10361-012 | For making bacmid DNA |
S.O.C. Medium | Thermo Fisher Scientific | 15544-034 | For DNA transformation |
CellFECTIN II Reagent | Thermo Fisher Scientific | 10362-101 | Insect cell transfection reagent |
Bac-to-Bac Expression System | Thermo Fisher Scientific | 10359-016 | Baculovirus-insect cells expression kit |
Bluo-gal | Thermo Fisher Scientific | 15519-028 | For isolation of recominant Bacmid DNA |
IPTG | Thermo Fisher Scientific | 15529-019 | For isolation of recominant Bacmid DNA |
pFastBac1 | Thermo Fisher Scientific | 10360014 | Baculorirus expression vector |
Sf9 cells | Thermo Fisher Scientific | 11496015 | Sf9 monolayer cells |
Hi5 cells | Thermo Fisher Scientific | B85502 | High Five insect cells |
Grace’s insect medium, unsupplemented | Thermo Fisher Scientific | 11595030 | Sf9 cell transfection minimum medium |
Grace’s insect medium, supplemented | Thermo Fisher Scientific | 11605102 | Sf9 monolayer cell culture complete medium |
Sf-900 II SFM | Thermo Fisher Scientific | 10902104 | Sf9 suspension cell culture medium without FBS |
Express Five SFM | Thermo Fisher Scientific | 10486025 | Hi5 cell culture medium |
Penicillin-Streptomycin | Thermo Fisher Scientific | 15140122 | 100 ml (10,000 I.U./ml) |
L-Glutamine (200 mM) | Thermo Fisher Scientific | 25030081 | 100 ml |
FBS Certified | Thermo Fisher Scientific | 16000-044 | 500 ml |
6-well plates | Thermo Fisher Scientific | 08-772-1B | Flat-bottom |
150 mm plates | Thermo Fisher Scientific | 353025 | 100/case |
1.5 ml Microcentrifuge Tubes | USA Scientific | 1415-2500 | 500 tubes/bag |
15 ml conical screw cap centrifuge tubes | USA Scientific | 1475-0511 | 25 tubes/bag |
50 ml conical screw cap centrifuge tubes | USA Scientific | 1500-1211 | 25 tubes/bag |
Ni-NTA Superflow | Qiagen | 30430 | NiNTA resin |
pH-indicator strips | EMD Millipore Corporation | 1.09535.0001 | pH 0 – 14 |