Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Podsumowanie

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

Streszczenie

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Wprowadzenie

The production of recombinant proteins using insect cell expression systems offers numerous benefits for the study of eukaryotic proteins. Namely, insect cells possess similar post-translational modifications, processing, and sorting mechanisms as those present in mammalian cells, which is advantageous for producing properly folded proteins^1,2,3. Insect cell systems also typically require fewer resources and less time and effort for maintenance than mammalian cell lines^4,5. The baculovirus expression system is one such insect cell-based system that is now widely used in many disciplines, including the production of recombinant proteins for protein characterization and therapeutics, the immunogenic presentation of foreign peptides and viral proteins for vaccine production, the synthesis of multi-protein complexes, the production of glycosylated proteins, etc.^1,2,4,6. There are, however, situations in which baculovirus expression may not be applicable^3,7, and the use of nonlytic and transient insect expression systems may be more appropriate. Specifically, transient insect cell expression offers the possibility for the rapid synthesis of recombinant protein, requires less development and maintenance, does not involve viral-imposed cell lysis, and provides a means to better study cellular trafficking during protein synthesis^7,8,9,10.

This protocol describes the rapid generation of expression vectors using two-step overlap extension PCR (OE-PCR) ¹¹ and the standard cloning of plasmid DNA in Escherichia coli. Plasmids are used to double-transfect commercially available cultured insect cells and to produce representative proteins. The protocol describes the production and use of two different fluorescently-labeled subcellular marker proteins and demonstrates colocalization with two aquaporin proteins from the insect Bemisia tabaci. The following protocol provides the basic methodology for OE-PCR, insect cell maintenance and transfection, and fluorescence microscopy for the cellular localization of target proteins.

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Protokół

1. OE-PCR for the Construction of Expression Plasmids

Note: See Table 1 for all primers used in OE-PCR. The use of a high-fidelity DNA polymerase is recommended for all amplifications. However, because these enzymes frequently do not leave a 3' A, it is necessary to perform a brief, non-amplifying incubation with a Taq DNA polymerase to "A-tail" the PCR products prior to cloning them into a TA insect cell expression vector. This protocol demonstrates a method to generate insect expression plasmids harboring chimeric proteins, with the fluorescent proteins fused in-frame to the carboxyl terminus of the genes of interest (in this case, to two Bemisia tabaci aquaporin proteins) or to subcellular marker proteins (Figure 1).

1. Use OE-PCR, which consists of two independent rounds of amplification, to generate: (1) sequences encoding the genes of interest (B. tabaci aquaporin 1: BtDrip1; B. tabaci aquaporin 2: BtDrip2_v1) fused in-frame with a green fluorescent protein variant called EGFP or (2) the subcellular markers (Drosophila melanogaster sex peptide receptor: DmSPR; Homo sapiens phospholipase A2: HsPLA2) with the mCherry coding sequence. Directly ligate the final OE-PCR products into the pIB/V5-His-TA plasmid.
   1. For the first round of OE-PCR (indicated by A-D and A'-D' in Figure 1), use a gene-specific sense primer (shown in bold in Table 1) and a chimeric antisense primer (italicized in Table 1) corresponding to the final 15 bp (without the stop codon) of the gene of interest/subcellular marker and to 15 bp of the 5'-end of the desired fluorescent protein (EGFP or mCherry) to generate a product with a 3' overhang.
      Note: See Table 2 for PCR conditions.
   2. In a separate tube, use a gene-specific fluorescent protein antisense primer (shown with an underline in Table 1) and a chimeric sense primer that contains 15 bp from the 3'-end of the gene of interest/subcellular marker and 15 bp from the 5'-end of the desired fluorescent protein to generate a product with a 5' overhang.
      Note: See Table 2 for the PCR conditions. The PCR template can either be a sequence-validated PCR product or, more preferably, plasmid DNA harboring the desired sequence. The study described here uses sequence-validated plasmids.
   3. Separate the PCR products with 1% agarose gel electrophoresis (using standard molecular-grade agarose).
   4. Excise and purify the products of the expected sizes (see Table 2) using a commercially available DNA gel extraction kit (see the table of materials for the specific kit used in this protocol).
   5. Use the gel-purified PCR products from step 1.1.4 to generate the final overlap extension products (indicated by A''-D'' in Figure 1). Use a gene-specific sense primer for the gene of interest/subcellular marker and the corresponding gene-specific antisense primer for the fluorescent protein to generate the desired chimeric sequence.
      Note: See Table 2 for the PCR conditions. It may be necessary to empirically determine the optimal amounts of first-round overlap extension products to add to the reaction mixture to yield the final, full-length chimeric PCR product.
   6. Separate the second-round OE-PCR products by 1% agarose gel electrophoresis. Excise and purify the products using a commercially available DNA gel extraction kit.
   7. Incubate gel-purified second-round OE-PCR products with 1 unit of Taq DNA polymerase master mix for 10 min at 72 °C to generate the 3' adenylated (i.e. A-tailed) products needed for TA cloning.
   8. Ligate the resulting adenylated products into the pIB expression vector and use standard molecular cloning techniques to transform chemically competent (heat shock) or electrocompetent (electroporation) Escherichia coli cells. Plate overnight on medium containing the appropriate selection marker (i.e., ampicillin or carbenicillin).
   9. Perform colony PCR¹² using a vector-specific primer and an insert-specific primer to identify insert-positive colonies that have been transformed with expression plasmids harboring an insert in the 5'-3' orientation. Prepare overnight cultures of colonies generating PCR products of the expected sizes.
   10. Isolate plasmid DNA using a commercially available DNA purification kit according to the manufacturer's instructions (see the Materials Table for the specific kit used in this protocol). Validate the sequence integrity of each insert by direct DNA sequencing.

2. Insect Cell Culture Maintenance

Note: To maintain sterile conditions, conduct all cell manipulations requiring the opening of the tissue culture flask within a laminar flow hood. Turn on the laminar flow hood UV germicidal lamp at least 1 h prior to cell manipulations. Wear nitrile gloves and decontaminate the surface of the bench, pipettes, utensils, tubes, and flasks with 70% ethanol before their use. Familiarity with basic cell culture techniques is recommended¹³.

1. Use Tni cells (an established cell culture line derived from Trichoplusia ni ovarian tissue) that are adapted to serum-free medium and maintain them as an adherent monolayer culture in serum-free insect medium at 28 °C in T25 tissue culture flasks.
2. Maintain Sf9 cells (an established cell culture line derived from Spodoptera frugiperda ovarian tissue) similarly but using TNM-FH insect culture medium supplemented with 10% Fetal Bovine Serum (FBS).
3. Seed the initial culture from frozen Sf9 or Tni cells by removing stock vials from -80 °C and allowing them to thaw in a 37 °C water bath. Decontaminate the vials with 70% ethanol after thawing and place them on ice.
4. Add 4 mL of insect cell medium to a new T25 flask and transfer 1 mL of the thawed insect cell suspension. Place the flask in a 28 °C, non-humidified incubator and allow the cells to attach for 30-45 min.
5. Replace the seeding medium with 5 mL of the appropriate medium and transfer the flasks to a 28 °C non-humidified incubator. Monitor cell confluence daily. Passage the cells when they reach 90% confluency.
   Note: Complete coverage of T25 flask corresponds to ~5 x 10⁶ cells.
6. Insect cell passage.
   1. To passage the cells, first remove the exhausted medium from the flask containing confluent cells using a sterile 5 mL serological pipette. Tilt the flask so that the medium flows to one corner, away from the cell monolayer. Carefully remove the medium using a pipette without disturbing the cells.
   2. Dislodge the Tni cells by gently rinsing the T25 flasks containing the confluent monolayer with 4 mL of serum-free insect medium using new, sterile, 5 mL serological pipette. Move the pipette tip across the flask and slowly irrigate to remove cells loosely attached to the flask bottom.
      1. Check for the adequate detachment of cells by removing all media, turning the flask over, and observing that the bottom of the flask is clear.
   3. For Sf9 cells, which adhere more tightly, add 4 mL of fresh TNM-FH medium and use a cell scraper to dislodge the attached cells. Use a 5 mL serological pipette to gently mix and reduce cell clumping.
   4. Use an automated cell counter to estimate the number of viable insect cells per volume of medium. Transfer 0.1 mL of cell/medium mixture to a 1.5-mL microfuge tube. In a separate 0.5 mL microfuge tube, add 10 µL of cell/medium mixture to 10 µL of trypan blue.
   5. Remove a cell counter chamber slide from its packaging and add 10 µL of the cell/medium/trypan blue mixture to each side of the counting slide. Insert the slide into the cell counter and determine the cell density and viability.
   6. Using the cell density, calculate and add the proper volume of insect cell medium (up to 5 mL) to new, sterile T25 flasks.
   7. Transfer approximately 1-1.5 x 10⁶ cells to T25 flasks with fresh media; label the flasks with the cell line, date, medium used, number of cells added, and passage number (P_n + 1 generation, where P_n is the passage number for the previous generation of cells); and place the flasks in a 28 °C incubator for up to 72 h.
      Note: Insect cells may be continuously propagated, although cells may be less receptive to transfection and/or heterologous protein expression after 30 passages. Treat all discarded cells/media with 10% bleach solution and autoclave disposable plastic ware before disposal.

3. Insect Cell Transfection

1. Seed a T25 flask with up to 1 x 10⁶ Tni or Sf9 cells in an appropriate insect cell medium (serum-free insect medium for Tni and TNM-FH for Sf9) and grow to confluency for 72 h at 28 °C.
2. Remove and discard the old medium and dislodge the cells with 4 mL of fresh serum-free insect medium (see steps 2.6.2 and 2.6.3, above).
3. Estimate the cell density using an automated cell counter (see step 2.6.4, above).
4. Add approximately 7 x 10⁵ cells to individual 35 mm glass-bottom dishes and allow the cells to attach for 20-25 min at 28 °C.
5. For each transfection, add 2 µg of plasmid DNA (either from one plasmid for single transfections or 2 µg from each of the two plasmids for double transfections) to 0.1 mL of serum-free insect medium (without FBS for both Sf9 and Tni transfections) in a sterile 1.5 mL microfuge tube.
6. In a separate tube, mix 8 µL of transfection reagent with 0.1 mL of serum-free insect medium and then transfer that solution to the tube containing the plasmid DNA of interest. Lightly vortex and incubate at RT for 20 - 30 min.
7. Dilute the plasmid-transfection mixture from steps 3.5 and 3.6 with 0.8 mL of serum-free insect medium so that the total volume equals 1 mL.
8. Carefully remove the media from the glass dishes containing attached cells. Overlay the attached cells with the diluted plasmid-transfection medium.
9. Incubate the cells at 28 °C for 5 h.
   Note: The conditions for transfection require empirical optimization for maximal transfection efficiency (e.g., overnight transfection rather than 5 h, the amount of plasmid DNA used, the chemistry of the transfection reagent used, etc.).
10. Remove and discard the transfection medium and gently wash the cells with 1 mL of serum-free insect medium, being careful not to dislodge cells.
11. Add 2 mL of fresh insect cell medium (serum-free insect medium for Tni and TNM-FH for Sf9) and incubate at 28 °C for 48-72 h.
    Note: Again, conditions require empirical optimization for maximal heterologous protein expression.

4. Confocal Fluorescence Microscopy

1. At 48-72 h post-transfection, wash the cells once with 1 mL of IPL-41 insect medium and then cover with 2 mL of IPL-41 for imaging.
   Note: This wash step reduces the background auto-fluorescence observed with insect cell medium used in normal cell maintenance.
2. Add 4 drops of Hoechst live-cell staining reagent (see the table of materials for the specific nuclear stain used in this protocol) to the medium and incubate at 28 °C for 20-25 min.
   Note: Additional nuclear stains may be substituted, although the dye should have a fluorescence profile unique from EGFP and mCherry.
3. Place a 35 mm dish into the self-enclosed laser scanning confocal microscope (see the table of materials for the specific instrument used in this protocol).
4. Adjust the microscope for Hoechst, EGFP, and mCherry observation conditions: Hoechst excitation/emission - 359/461 nm; EGFP excitation/emission - 489/510 nm; mCherry excitation/emission - 580/610 nm.
5. Perform an initial scan using a 10x objective to confirm fluorescent expression and then switch to scanning mode using a 60X phase contrast water-immersion objective.
6. Adjust the laser power (5-7%), detector sensitivity (47-49%), scanning speed, Z-axis depth, and digital zoom to optimize the image contrast and resolution. Image the cells at 1.5X digital zoom to give a total of 90X amplification.
   Note: Microscope parameters require empirical adjustment for optimal image collection and may be specific to the instrument in use.
7. Export the raw data as TIFF image files and modify (crop and overlay) for figure generation.

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Wyniki

OE-PCR

OE-PCR allows for the synthesis of chimeric DNA products that, once inserted into an expression vector, allow for the production of recombinant chimeric proteins corresponding to any test gene of interest and fluorescent marker protein. Figure 1 represents a general scheme for the production of pIB expression vectors containing B. tabaci aquaporin coding sequences (BtDrip1 and BtDrip2_v1) in-frame w...

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Dyskusje

Heterologous protein expression systems are important tools for the production of recombinant proteins used in numerous downstream applications⁴. Choosing from the diverse expression systems available depends on the end goal for the protein of interest. Several insect cell expression systems are available that offer flexible alternatives to prokaryotic and eukaryotic cell expression systems^5,6. Insect systems requiring baculovirus infectio...

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Materiały

Name	Company	Catalog Number	Comments
KOD DNA Polymerase	EMD Millipore	71085-3	High-fidelity DNA polymerase used for PCR amplification of overlap extension PCR products
ExTaq DNA Polymerase	TaKaRa-Clontech	RR001B	DNA polymerase used for A-tailing of PCR products
EconoTaq PLUS GREEN 2x DNA Polymerase Master Mix	Lucigen	30033-1	DNA polymerase used for bacterial colony PCR
Biometra TProfessional Gradient Thermocycler	Biometra/LABRepCo	070-851
Agarose LE	Benchmark Scientific	A1705
SYBR Safe DNA Gel Stain	ThermoFisher	S33102
Montage DNA Gel Extraction Kit	EMD Millipore	LSKGEL050
pIB/V5-His TOPO TA Expression Kit	ThermoFisher	K89020	Contains components needed to clone overlap extension PCR products, including linearized and topoisomerase I-activated pIB/V5-His-TOPO vector, One Shot TOP10 chemically competent E. coli, and salt solution.
QIAprep Spin MiniPrep Kit	Qiagen	27104
QIAcube Robotic Workstation	Qiagen	9001292
Purifier Vertical Clean Bench	Labconco	3970401
Tni cultured insect cell Line	Allele Biotech	ABP-CEL-10005
Sf9 cultured insect cell Line	Allele Biotech	ABP-CEL-10002
Serum-Free Insect Culture Medium	Allele Biotech	ABP-MED-10002
TNM-FH Insect Culture Medium	Allele Biotech	ABP-MED-10001
IPL-41 Insect Medium	ThermoFisher	11405081
Cellfectin II Transfection Reagent	ThermoFisher	10362100
16 cm Disposable Cell Scrapers	Sarstedt	83.1832	Cell scrapers with two-position blade
25 cm2 (T25) Tissue Culture Flasks with Vent Filter Caps	Life Science Products	CT-229331
Transfer Pipets	Fisher	1371120
Sterile, 50 mL Bio-Reaction Tubes	Life Science Products	CT-229475
PipetteBoy	VWR	14222-180
5 mL Serological Pipettes	Sarstedt	86.1253.001
0.5 mL Flat-Cap PCR Tubes	Fisher	14230200
Polypropylene Biohazard Autoclave Bags	Fisher	01828C
35 mm #1.5 Glass Bottom Dishes	Matsunami Glass	D35-14-1.5-U	35 mm dish diameter, 14 mm glass diameter, 1.5 mm glass thickness, uncoated
Incubator, Model 1510E	VWR	35823-961
Countess II FL Cell Counter	ThermoFisher	AMQAF1000
Countess Cell Counting Chamber Slides with 0.4% Trypan Blue Reagent	ThermoFisher	C10228
Fluoview FV10i-LIV Laser Scanning Confocal Microscope	Olympus	FV10i-LIV
HsPLA2/pCS6 plasmid DNA	transOMIC Technologies	TCH1303
pmCherry Vector	Clontech	632522
NucBlue Live ReadyProbes Reagent (Hoechst 33342)	ThermoFisher	R37605