LEXSYcon Expression Kit• The LEXSinduce kit (EGE-1200) contains one of the pLEXSY_I vectors (EGE-120 and EGE 121) for tetracycline-inducible bacteriophage-T7-based expression in

LEXSYcon Expression Kit contains any of the pLEXSY vectors

pLEXSY-sat (Cat.-No. EGE-204)

pLEXSY-ble (Cat.-No. EGE-201)

pLEXSY-hyg (Cat.-No. EGE-202)

pLEXSY-neo (Cat.-No. EGE-203)

User’s Guide

Cat.-No.: EGE-1000

General purpose:

FOR RESEARCH USE ONLY!

NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USE!

Jena Bioscience GmbH Loebstedter Str. 80

07749 Jena, Germany Tel.: +49-3641-6285000 Fax.: +49-3641-6285100

Email: [email protected]

Jena Bioscience GmbH • Loebstedter Str. 80 • D-07749 Jena • Germany • e-Mail: [email protected]

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1. INTRODUCTION 3

2. KIT COMPONENTS AND STORAGE CONDITIONS 5

2.1. LEXSY host P10 5

2.2. pLEXSY expression vector 5

2.3. Primers for diagnostic PCRs and sequencing 5

2.4. Ingredients for 1 liter of culture medium 6

2.5. Equipment and materials supplied by customer 6

3. CULTIVATION OF LEXSY HOSTS AND EXPRESSION STRAINS 7

3.1. Preparation of growth medium 7

3.2. Cultivation conditions 7

3.3. Storage of LEXSY host and recombinants - Glycerol stocks preparation 8

3.4. Glycerol stocks reactivation, initial cultivation 8

4. ENGINEERING OF LEXSY EXPRESSION STRAINS 9

4.1. Amplification of target gene 9

4.2. Insertion of target gene into pLEXSY expression vector 11

4.3. Preparation of plasmid for LEXSY host transfection 11

4.4. Transfection of LEXSY host strains by electroporation 12

5. SELECTION OF TRANSGENIC LEXSY STRAINS 13

5.1. Clonal selection by plating on solid media 13

5.2. Selection in suspension culture 13

5.3. Confirmation of genomic integration by diagnostic PCR 14

5.4. Evaluation of target protein expression level 15

6. LICENSING INFORMATION 16

7. LITERATURE 16

8. APPENDIX 17

8.1. Sequences of the primers available for LEXSY 17

8.2. Preparation of LEXSY BHI agar plates for clonal selection 19

8.3. Alternative electroporation: High-Voltage protocol for transfection of LEXSY 20

8.4. How to grow a Leishmania culture 21


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1. Introduction LEXSY – the unique protein expression system offered by Jena Bioscience combines the potential of eukaryotic protein expression/folding/modification with easy handling known from bacterial expression systems. The trypanosomatid protozoan host Leishmania tarentolae used in LEXSY was first isolated from the Moorish gecko Tarentola mauritanica, is kept in axenic culture, and is not pathogenic to mammals (Biosafety level 1).

In trypanosomatid protozoa mRNAs are transcribed as polycistronic precursors that are posttranscriptionally processed by trans-splicing and polyadenylation within the intergenic regions into individual mRNAs (1). Regulation of protein expression in these species occurs mainly on the level of RNA and may be influenced by the structure of the intergenic regions (1, 2). For the expression of heterologous proteins in L. tarentolae we have optimized splicing signals (3).

For high-level expression target genes are supplied with specific splicing signals provided on the expression vector included in the kit and stably integrated into a chromosomal locus or maintained episomally. The expression vectors are constructed in E. coli and introduced into the LEXSY hosts by electroporation.

Jena Bioscience offers three types of LEXSY Kits, and these contain all components for engineering and selecting expression strains:

• The LEXSYcon kit (EGE-1000) contains one of the pLEXSY vectors (EGE-201 to EGE-204) for constitutive expression in the cytoplasm of the LEXSY host P10.

• The LEXSecrete kit (EGE-1100) contains one of the pLEXSY_S vectors (EGE-211 to EGE-214) for constitutive secretory expression in the LEXSY host P10.

• The LEXSinduce kit (EGE-1200) contains one of the pLEXSY_I vectors (EGE-120 and EGE-121) for tetracycline-inducible bacteriophage-T7-based expression in the LEXSY host T7-TR.

Recombinant LEXSY strains can be cultivated as static or agitated suspension cultures. They grow to high cell densities (5x108 cells/ml) in complex LEXSY media (LEXSY BHI or LEXSY YS). The cells can be easily lysed in a microfluidizer, by sonication, osmotic shock or detergents.

Numerous cytosolic, membrane-localized and extracellular proteins have been expressed with LEXSY at Jena Bioscience with yields up to 300 mg per liter in suspension cultures. In addition, we have

Fig. 1: Transcription in trypanosomatid protozoa. Fig. 2: LEXSY cells expressing green-fluorescent protein


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established growth of LEXSY strains in laboratory fermentation plants with volumes of 1 – 30 liter. For further details, please, visit our website http://www.jenabioscience.com.

The LEXSYcon kit contains any of the four expression vectors pLEXSY-sat (encoding streptothricine acetyltransferase), pLEXSY-hyg (encoding hygromycin phosphotransferase), pLEXSY-ble (encoding bleomycin resistance gene), or pLEXSY-neo (encoding aminoglucoside phosphotransferase) allowing selection with the antibiotics LEXSY Ntc (Nourseothricin), LEXSY Hygro (Hygromycin), LEXSY-Bleo (Bleomycin) or LEXSY Neo (Neomycin), respectively.

One main advantage of the LEXSY Systems is the potential for mammalian-type posttranslational modification of target proteins, such as glycosylation (see Fig. 3), phosphorylation or prenylation. Recombinant human Erythropoietin (EPO) purified at Jena Bioscience using the LEXSecrete kit was biologically active, natively processed at the N-terminus, and N-glycosylated (3). The N-glycosylation profile was exceptionally homogenous, with a biantennary oligosaccharide and the Man3GlcNAc2 core structure accounting for >90% of the glycans present. L. tarentolae is thus the first described biotechnologically useful unicellular eukaryotic organism producing biantennary, fully galactosylated, core-α-1,6-fucosylated N-glycans. This N-glycosylation profile was coincident with the profile of recombinant human Interferon-γ expressed in LEXSY and of LEXSY host Gp63 glycoprotein.

Fig. 3: N-glycosylation of proteins in LEXSY hosts, compared to glycosylation in other protein expression systems. Note that the glycosylation pattern obtained in mammalian cells and in LEXSY only differs in the presence of N-acetylneuraminic acid at the ends of the sugar chains in the latter.


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2. Kit components and storage conditions The kit is shipped as two parcels. Parcel 1 contains the frozen glycerol stocks of the LEXSY host strain and is shipped on dry ice. Parcel 2 contains all other kit components and is shipped on pre-cooled gel packs maintaining a temperature of 4 to 15°C. Short exposure of these components to ambient temperature during shipment will not interfere with the performance of the kit.

2.1. LEXSY host P10

The kit contains 3 vials with 1.6 ml each of frozen glycerol stocks of LEXSY host P10. These stocks can be stored at -80°C for at least 1 year. For reactivation refer to section 3.

2.2. pLEXSY expression vector

one of the vectors

pLEXSY-sat

pLEXSY-hyg

pLEXSY-ble

pLEXSY-neo

− 5 µg in 10 mM TrisHCl pH 8.0 − store at -20°C − stable for at least 2 years

2.3. Primers for diagnostic PCRs and sequencing

one of the vector-specific primers

sat forward primer F2999, for pLEXSY-sat

hyg forward primer A3804, for pLEXSY-hyg

ble forward primer A708, for pLEXSY-ble

neo forward primer A1432, for pLEXSY-neo

plus the primers

Insert sequencing forward primer A992

Insert sequencing reverse primer A264

F4 reverse primer A1045

ssu forward primer F3001

ssu reverse primer F3002

− 50 µM in 10 mM TrisHCl pH 8 − store at -20°C − stable for at least 2 years


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2.4. Ingredients for 1 liter of culture medium

LEXSY BHI, Powder

− 37 g − store at ambient temperature − stable for at least 3 years

one of the antibiotics

LEXSY NTC (Nourseothricin), for pLEXSY-sat, ready-to-use 1000x stock solution, 100 mg/ml

LEXSY Hygro (Hygromycin), for pLEXSY-hyg, ready-to-use 1000x stock solution, 100 mg/ml

LEXSY Bleo (Bleomycin), for pLEXSY-ble, ready-to-use 1000x stock solution, 100 mg/ml

LEXSY Neo (Neomycin), for pLEXSY-neo, ready-to-use 1000x stock solution, 50 mg/ml

− 1 ml − filter sterilized − store at -20°C − stable for 12 months

Additives

Hemin, Stock solution, 500x (0.25% solution of porcine Hemin in 50% Triethanolamine)

− 2 ml − filter-sterilized − store at 4°C in the dark − stable for at least 12 months

Pen-Strep, Stock solution, 200x, contains 10.000 units of penicillin (base) and 10.000 µg of streptomycin (base)/ml as penicillin G sodium salt and streptomycin sulfate in 0.85% saline

− 5 ml − filter-sterilized − store at -20°C − stable for at least 12 months

Pen-Strep may be added for avoiding growth of bacterial contaminants, but is, in contrast to Hemin, no prerequisite for LEXSY growth

2.5. Equipment and materials supplied by customer

Incubator at 26°C

Inverse Microscope (or standard Microscope)

Electroporation device, e.g. BioRad GenePulser II with capacity extender or GenePulser Xcell with PC and CE Modules

Cooling and freezing capacities at +4°C, -20°C and -80°C

Standard molecular biology equipment for PCR and cloning


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3. Cultivation of LEXSY hosts and expression strains

LEXSY hosts and expression strains can be cultivated in the dark at 26°C in complex media (LEXSY BHI or LEXSY YS) or chemically defined media (Synthetic LEXSY medium), both supplemented with Hemin, which is essential for Leishmania. There is no need to add sera to complex media. Addition of fetal calf serum will not enhance growth of L. tarentolae in complex media. To prevent bacterial infections, Penicillin and Streptomycin (Pen-Strep) may be added.

If you encounter growth problems with the host or LEXSY strains, centrifuge cells 5 min at 2000 x g, resuspend pellet carefully in fresh medium and continue incubation.

3.1. Preparation of growth medium

Dissolve 37 g/l LEXSY BHI powder in deionized water and autoclave 15 min at 121°C. Store at room temperature.

Add to 200 ml LEXSY BHI medium

− 1.0 ml Pen-Strep stock solution, 200x − 0.4 ml Hemin stock solution, 500x (final concentration 5 µg/ml).

3.2. Cultivation conditions

L. tarentolae needs aerobic conditions for development. The strain can be maintained as continuous suspension culture with regular dilutions at 1:10 to 1:50 rates. Best results are obtained with inoculations during mid-late growth phase (OD 2-3). Avoid inoculation from late-stationary phase cultures.

Cultivation may be performed in

• ventilated tissue culture flasks for suspension cultures, culture volume 10 to 200 ml

• Erlenmeyer flasks, agitated in an incubator at approx. 170 rpm, culture volume of 50 ml to 1 liter

• standard bioreactors, up to 100 liter.

Selection of recombinants:

• vector pLEXSY-sat: 100 µg/ml LEXSY NTC (Nourseothricin)

• vector pLEXSY -hyg: 100 µg/ml LEXSY Hygro (Hygromycin)

• vector pLEXSY -ble: 100 µg/ml LEXSY Bleo (Bleomycin)

• vector pLEXSY-neo: 50 µg/ml LEXSY Neo (Neomycin)


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3.3. Storage of LEXSY host and recombinants - Glycerol stocks preparation

The LEXSY host and LEXSY strains may be stored at -80°C in 20% glycerol for at least one year. We even recovered viable cells under these conditions after 9 years of storage, without any loss of vitality.

• Add 1.2 ml autoclaved Glycerol (80%) to 15 ml Falcon tube

• Withdraw 3.6 ml of culture grown in LEXSY BHI* medium (Cat.-No. ML-411) from mid growth phase 4-8 x 107 cells/ml (OD 1.2-1.8)

• Mix with glycerol and distribute 3 x 1.6 ml each to sterile cryovials

• keep 10 min at room temperature

• keep 1 h on wet ice

• keep o/n at –20°C

• transfer to –80°C for long term storage

3.4. Glycerol stocks reactivation, initial cultivation

• Thaw frozen glycerol stock on ice

• Inoculate the entire content of the vial into 10 ml of LEXSY BHI* medium with appropriate antibiotic(s) Motile cells can be observed immediately after inoculation by microscopy

• Incubate as static suspension culture in ventilated TC flask at 26°C for 2 days

• Dilute dense culture 1:10 into fresh LEXSY BHI* and incubate for 3 days

• For strain maintenance dilute 1:20 into fresh LEXSY BHI* on Monday and Friday each week

* contains Hemin and PenStrep


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4. Engineering of LEXSY expression strains This kit was designed for constitutive expression of target proteins following integration of the expression cassette into the chromosomal 18S rRNA locus (ssu) making use of the high level transcription rates by the host RNA polymerase I. In the first step the ORF for the target gene is supplied with linker sequences containing restriction sites allowing insertion into the pLEXSY vectors.

The pLEXSY vectors contain optimized nontranslated regions flanking the target gene insertion site and provide the splicing signals for posttranscriptional mRNA processing. Following construction in E. coli the expression plasmid is linearized and integrated into the ssu locus of the LEXSY host by homologous recombination (double crossover). There are no signals for transcription and/or translation in E. coli preceding the target gene insertion site, and lack of gene expression in E. coli was demonstrated with a reporter gene. This is of advantage for the generation of constructs for proteins toxic for E. coli.

4.1. Amplification of target gene

The target gene may be inserted into the expression site of pLEXSY vectors (Fig. 4) as BglII or NcoI or XhoI (SlaI) x XhoI (SlaI) or PacI or NotI compatible cassette. The most convenient approach is to generate a NcoI x NotI cassette where the NcoI site overlaps the ATG codon of the target gene and the NotI site is preceded by the stop codon.

• Analyse your target gene for internal BglII, NcoI, XhoI (SlaI), PacI or NotI sites. If possible choose to generate a NcoI x NotI cassette. Be aware that PciI and BspHI ends are compatible with NcoI ends, as are BamHI and BclI ends with BglII ends and SalI ends with XhoI (SlaI) ends. This consideration adds to the insertion variability. The cloning sites of pLEXSY vectors will allow you to select the appropriate restriction sites not present on your gene for insertion into the expression vector.

Make sure your target gene does not contain any internal SwaI site, since this site will be used for vector linearization. If a Swa I site is present, however, you may choose to remove it prior to cloning by silent mutagenesis or to transfect circular DNA (ref. to 6. 2.).

• Design a forward and reverse primer pair for amplification of your target gene with linker sequences containing the selected restriction sites allowing integration into the pLEXSY vector. BclI will work on PCR fragments since the DNA is not methylated. If you don´t use NcoI it is recommended to maintain the sequence of the BglII – NcoI region preceding the start codon (Fig. 1) as close as possible. The triplet preceding the ATG seems to be important for the expression level of the target gene. ACC, CCC, GCC or ATC were favourable with a reporter gene. However, there is no evidence, that this is true for any gene. GGG, GGT, TAA, TAC or ATG were not found so far at this position in Leishmania.

• Amplify the target gene by standard PCR techniques with a high fidelity polymerase, gel-purify the fragment and trim the ends with the appropriate restriction enzymes (e.g. NcoI x NotI). Optionally, you may subclone the PCR fragment e.g. in a TOPO (Invitrogen), pJET (Fermentas) or pGEM (Promega) vector and generate the gene cassette by restriction of the resulting plasmid. This has the advantage that cleavage by both enzymes can be monitored and sequence confirmation can be performed prior to insertion into the expression vector. Prepare the target gene fragment for ligation.


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BglII NcoI XhoI XhoI PacI NotI AGATCTGCCATGGCTCGAGnnnnnnnnnCTCGAGTTAATTAAGCGGCCGC TCTAGACGGTACCGAGCTCnnnnnnnnnGAGCTCAATTAATTCGCCGGCG

Fig. 4:

Map of the pLEXSY vectors and cloning sites for the target gene replacing the 0.7 kb stuffer fragment. Instead of a polylinker, the target gene replaces a stuffer fragment, allowing control for proper restriction. 5´ssu and 3´ssu are regions for homologous recombination into the host chromosome following linearization of the expression plasmid with SwaI. 0.4k-IR cam BA, 1.4k-IR cam CB and 1.7k-IR are optimized gene-flanking nontranslated regions providing the splicing signals for posttranscriptional mRNA processing for expression of target and marker genes in the LEXSY host P10. Numbering indicates the positions of restriction sites in pLEXSY-sat.

marker

pLEXSY vector


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4.2. Insertion of target gene into pLEXSY expression vector

The pLEXSY vectors allow directional insertion of the target gene cassette by replacement of a stuffer fragment (Fig. 4). This is advantageous to insertion into a singular multiple cloning site since vector cleavage by both restriction enzymes can be monitored by the appearance of the 0.7 kbp stuffer fragment.

• Digest the expression vector provided in the kit with the appropriate restriction enzymes (e.g. NcoI x NotI)

• Gel-isolate the larger, 7 - 8 kbp fragment and prepare it for ligation.

• Ligate the vector and the target gene fragment with T4 DNA ligase (e.g. JBS Cat.-No. EN-149) using standard ligation methods, and transform competent E. coli cells with the ligation mix. Include controls for vector linearization, ligation and transformation. Since pLEXSY vectors encompass repetitive and homopolymeric DNA stretches within the nontranslated regions (utr), it is recommended to use E. coli XL10, DH5α (Stratagene), Stbl2 or Stbl4 (Invitrogen) as recipient strains.

• Select recombinant E. coli clones with ampicillin and screen for the presence of the insert in the plasmids. Directional cloning into pLEXSY vectors normally yields high rates of positive clones. Insert screening may be performed by restriction analysis of recombinant plasmids isolated from a small number of cultures grown o/n in 1 - 3 ml LB with ampicillin at 30°C.

• Prepare at least 5 µg plasmid DNA from a positive clone for digestion and sequence confirmation and subsequent transfection. Usually, it is sufficient to isolate plasmid DNA with a commercial kit from 10 ml o/n culture grown at 30°C.

• Confirm plasmid identity by restriction and sequence analysis of the insert and of the vector/insert fusions using the forward A992 and reverse A264 primers included in the kit. Both primers are proved for function in cycle sequencing protocols. Forward primer A992 (calculated Tm = 58°C) anneals 5´of the insert, reverse primer A264 (calculated Tm = 46°C) 3´ of the insert of pLEXSY vectors. The primer sequences are shown in the appendix.

4.3. Preparation of plasmid for LEXSY host transfection • Be sure, that the target gene does not contain a SwaI site (If so you may use circular plasmid

for transfection. However, this may lead to lower expression levels from episomal DNA).

• Digest to completion 1 - 5 µg of the obtained expression plasmid containing the target gene with SwaI or the isoschizomer SmiI. This treatment will generate a 2.9 kbp fragment representing the E. coli part and a larger fragment representing the linearized expression cassette with the target gene to be integrated into the ssu locus of the LEXSY host.

• For best performance, gel-isolation of the expression cassette is recommended. Gel isolation of the fragment to be transfected into the LEXSY host may be done with any commercial kit, e.g. the JBS Agarose Gel Extraction Kit (Cat.-No. PP-202).

• If the digested plasmid is used without fragment purification, enzymes and buffer salts may be removed by using the JBS PCR Purification Kit (Cat.-No. PP-201). Alternatively, precipitate the digest with ethanol, wash with 70% ethanol and redissolve at 0.5 µg/µl in sterile double distilled water or 10 mM Tris pH 8. Control the digest and the concentration by gel electrophoresis. This preparation is now ready for transfection.


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4.4. Transfection of LEXSY host strains by electroporation

For efficient transfection it is recommended to continuously grow LEXSY host cultures by successively transferring the strains to fresh medium at 1:10 to 1:50 dilutions once or twice a week.

Prior to transfection calibrate the OD readings of your spectrophotometer at a defined wavelength between 550 and 600 nm to the cell densities of the LEXSY host strain cultures by taking OD readings and counting cells immortalized in 3% formalin (e.g. 1:10 dilutions in a Neubauer chamber) at different time points during growth of suspension culture. Dependent on the spectrophotometer and wavelength used, this correlation may be different from the data specified here.

• Inoculate L. tarentolae preculture 1:20 in 10 ml LEXSY BHI* medium (Cat.-No. ML-411) on Friday afternoon and incubate in tissue culture (TC) flask upright @ 26°C until Monday

• On Monday afternoon dilute preculture 1:10 in 10 ml LEXSY BHI* medium and incubate in TC flask flat @ 26°C o/n

• Grow culture to 6 x 107 cells/ml (OD 1.4); ensure by microscopy that the cells are vital and of droplike shape

• Spin cells 5 min, 2000xg at room temperature and remove ½ volume of supernatant

• Resuspend pellet in remaining medium (108 cells/ml) and put on wet ice for 10 min

• Have ready on wet ice in parallel tubes with 0.1-5 µg transforming DNA in 50 µl water and electroporation cuvettes d=2 mm

• Add 350 µl precilled cells to the tube with DNA, mix and transfer to the electroporation cuvette on wet ice

• Electroporate @ 450V, 450µF and monitor pulse time (5-6 msec)

• Put cuvette back on ice for exactly 10 min (timer)

• Transfer electroporated cells with capillary to 10 ml LEXSY BHI*

• Incubate o/n at 26°C (ca. 20h, OD 0.3-0.4)

• Spin batches of 2 ml of o/n culture 5 min, 2000xg, resuspend pellets in 50-100 ml residual medium and plate on BHI-agar with selection antibiotic(s) and/or continue selection in suspension culture

• After 5-7 days count colonies and/or dilute suspension cultures 1:10

• Expand colonies in 0.2 ml in 96 well 1 ml in 24 well format 10 ml TC flask

* contains Hemin and PenStrep


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5. Selection of transgenic LEXSY strains For establishment of expression cultures we routinely use the two methods described below in parallel. We repeatedly found similar expression levels when comparing cultures derived from a clonal selection by plating on solid media (5.1.) with suspension cultures (5.2.).

5.1. Clonal selection by plating on solid media

Plating of cells after transfection allows selection of genetically defined, recombinant clones. For plating, LEXSY BHI agar plates are always freshly prepared as described in the appendix, 8.2. Everything required for preparing LEXSY BHI agar plates is included in our LEXSY Plating Kit (Cat.-No. ML-451).

• Withdraw 1 - 4 batches of 2 ml from the 10 ml o/n culture before first addition of antibiotic. Pellet cells for 5 min at 2000g and 20°C.

• Decant the supernatant and resuspend cells in 50-100 µl residual medium.

• Carefully plate resuspended cells onto freshly prepared LEXSY BHI agar plates supplemented to the recommended final concentration with the respective LEXSY antibiotic (e.g. Nourseothricin for pLEXSY-sat; see 3.2.). The remaining culture may be used for selection in suspension culture as described below (see 5.2.).

• We routinely streak the cells onto nitrocellulose filters placed on the agar plates. Plating is easier on these membranes than on the 1% agar, and swarming of cells, as observed on agar, is drastically diminished.

• 5 – 7 days after plating small, defined colonies begin to appear on a slight background lawn.

• After these colonies have grown up to 1 – 2 mm diameter (approx. 7 – 9 days), transfer them to 0.2 ml of selective growth medium into wells of a 96-well plate using a pipette tip.

• After 24 hours expand these clones into 1 ml selective medium in 24-well plates, and another 24 – 48h later transfer to 10 ml medium in TC flasks.

5.2. Selection in suspension culture

As soon as the 10 ml o/n cultures obtained in the transfection experiments (see 4.4.) start to get slightly turbid (OD600 0.4, ca. 107 cells/ml; usually approx. 20 h after electroporation), add the appropriate LEXSY antibiotic (see 3.2) to the recommended final concentration from the filter-sterilized stock provided in the kit and continue incubation at 26°C. Don´t let the cultures overgrow before selection, since it will take longer to kill non-recombinant cells!

• Follow the status of the cultures microscopically and visually until you start seeing the difference to the cells electroporated without DNA under the same conditions (negative control). Recombinant cells are motile, of droplike shape and grow as a “cloudy” suspension culture whereas the cells in the negative control begin to die during the selection period and appear as spherical or irregular forms without flagella under the microscope. Visually, however, the negative control may also appear as a turbid suspension.

• It usually takes one consecutive transfer into fresh medium with appropriate antibiotic at 1:10 inoculation rate to get a turbid culture of antibiotic-resistant, recombinant cells and a clear negative control (absence of growth). This passage should be performed within 7 days after first drug addition. Do not wait for a longer period to passage the culture even if the negative control also got turbid.

• If at the first addition of selective antibiotic the cultures already were too dense, the negative control may not be dead after the first 1:10 passage. In this case another (or even more) subsequent 1:10 dilution into fresh medium with antibiotic may be needed to get a turbid recombinant culture and a clear negative control.


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5.3. Confirmation of genomic integration by diagnostic PCR

Integration of the expression cassette into the ssu locus can be confirmed by diagnostic PCR of genomic DNA of transgenic strains. For this purpose primer pairs including one primer hybridizing within the expression cassette and one primer hybridizing to a ssu sequence not present on the plasmid are used in diagnostic PCR. A set of primers for such diagnostic PCRs and for insert sequencing is included in each kit. The sequences are found in the appendix of the manual (see 8.1.).

Prepare genomic DNA from 2 ml dense culture (OD approx. 3) by conventional phenol/chloroform extraction or with a commercial kit (e.g. DNeasy Tissue DNA isolation Kit, QIAGEN Cat.-No. 69504; follow the protocol for Gram-negative bacteria of this kit. There is, however, no need to add protease).

For pLEXSY vectors: Perform diagnostic PCR (annealing temperature 53°C) with the antibiotic resistance cassette forward primer and ssu reverse primer F3002 included in the kit. Integration of the expression cassette into the ssu locus will result in a characteristic fragment for each vector, which is not observed in control reactions where the template is the expression plasmid or genomic DNA from the LEXSY host. In addition you may perform diagnostic PCR (annealing temperature 60°C) with ssu forward primer F3001 and F4 reverse primer A1045 (hybridizing within 5´utr of target gene) included in the kit. Integration of the expression cassette into the ssu locus will yield a characteristic 0.95 kbp fragment not obtained in control reactions where the template is the expression plasmid or genomic DNA from the LEXSY host strain.

Additional diagnostic PCR reactions including target gene-specific primers may be performed.


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5.4. Evaluation of target protein expression level

The expression level of the target protein in recombinant LEXSY strains may be evaluated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting of cell extracts or, in case of secretory expression, aliquots from supernatants. For obtaining optimal expression of each individual protein, we recommend to check different cultivation conditions. We also recommend checking for the presence of the recombinant protein in both, the cells and the medium, irrespective of the expression plasmid used.

• Inoculate 10 - 200 ml LEXSY BHI supplemented with Hemin + Pen-Strep and the respective LEXSY antibiotic, 1:10 to and grow at 26°C as agitated cultures in Erlenmeyer flasks shaking at approx.170 rpm.

• Take aliquots from different time points of cultivation (e.g. OD 1 - 4) for estimation of optimum harvest time.

• For both, cell extracts and supernatants analysis calculate volume of aliquot V [ml] = 2/OD, e.g.

o withdraw 4 ml culture @ OD 0.5

o withdraw 2 ml culture @ OD 1.0

o withdraw 1 ml culture @ OD 2.0 etc.

• Sediment cells 5 - 10 min at 2.000g.

For testing secretory protein expression concentrate culture supernatants 50 - 200x with 10% trichloroacetic acid (TCA) as follows:

• Collect the supernatant and add ice-cold TCA to a final concentration 10%.

• Leave on ice for 30 min, then spin 15 min 15.000g at 4°C.

• Discard supernatant, and resuspend pellet in 80% acetone to remove residual TCA.

• Spin 15 min 15.000g at 4°C, dry and resuspend pellet in a final volume of 10 - 50 µl 2x SDS PAGE loading buffer

• Apply 10 - 20 µl sample/slot for SDS-PAGE and Western blotting.

For testing cellular expression levels, the cell pellets are used.

• Lyse pellets in 0.1 - 0.2 ml 2x gel-loading buffer

• Apply 10 - 20 µl sample/slot for SDS-PAGE and Western blotting


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6. Licensing information Purchase of the LEXSY Expression Kits includes a non-exclusive and non-transferable license for non-commercial research. Commercial use of the LEXSY expression system, however, requires separate licensing.

Commercial use includes but is not limited to:

• the use of any protein or other substance produced by LEXSY as reagents in screening to discover and/or promote candidate compounds for sale to a customer, distributor, wholesaler or other end user in therapeutic, diagnostic, prophylactic, and/or veterinary areas.

• the manufacture, sale or offer to sell of any product containing proteins or other substances produced by LEXSY.

• the large-scale production of recombinant protein pharmaceuticals.

• "Contract research" to any third party or "Contract manufacturing" for any third party that has not been granted a license to use LEXSY.

Please contact us at [email protected].

7. Literature (1) Teixera SMR (1998) Braz. J. Med. Biol. Res. 31: 1503-1516

(2) Clayton CE (2002) EMBO J. 21: 1881-1888

(3) Breitling R, Klingner S, Callewaert N, Pietrucha R, Geyer A, Ehrlich G, Hartung R, Müller A, Contreras R, Beverley S and Alexandrov K (2002) Prot. Expr. Purific. 25: 209-218

(4) Robinson KA & Beverley SM (2003) Mol. Biochem. Parasitol. 128: 217-228

(5) Beverley SM, Clayton CE (1993) Methods in Molecular Biology, Vol.21: Protocols of Molecular Parasitology chapter 25 p. 333-347


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8. Appendix

8.1. Sequences of the primers available for LEXSY

sat forward primer F2999

integration diagnostics of all sat expression vectors

5’-CCTAGTATGAAGATTTCGGTGATC-3’ PM-103

ssu reverse primer F3002

integration diagnostics of all ssu integration vectors

5’-CTGCAGGTTCACCTACAGCTAC-3’ PM-104

ssu forward primer F3001

integration diagnostics of all sat expression vectors

5’-GATCTGGTTGATTCTGCCAGTAG-3’ PM-103

F4 reverse primer A1045

integration diagnostics of all ssu integration vectors

5’-CATTACGTCGCACGTTATCG-3’ PM-105

Insert sequencing forward primer A992

all "F4" expression vectors with 5'utr camBA

5’-CATCGAAGGGATTCATCGC-3’ PM-102

Insert sequencing reverse primer A264

all expression vectors

5’-CATCTATAGAGAAGTACACGTAAAAG-3’ PM-101

ble forward primer A708

integration diagnostics of all ble expression vectors

5’-GGATCCACCGCATGGCCAAGTTGACCAGTG-3’ PM-107

neo forward primer A1432

integration diagnostics of all neo expression vectors

5’-GCATGGCGATGCCTGCTTGC-3’ PM-108

hyg forward primer A3804

integration diagnostics of all hyg expression vectors

5’-CCGATGGCTGTGTAGAAGTACTCG-3’ PM-109

Insert sequencing forward primer P1442

all "AP" expression vectors with 5'utr aprt

5’-CCGACTGCAACAAGGTGTAG-3’ PM-110

AP reverse primer A1715

integration diagnostics of all "AP" expression vectors with 5'utr aprt

5’-TATTCGTTGTCAGATGGCGCAC-3’ PM-111

tub forward primer E158

integration diagnostics of all β-tub integration vectors

5’-CCGGCCAGTGCGGCAACCAGATCGG-3’ PM-112

tub reverse primer E159

integration diagnostics of all β-tub integration vectors

5’-GGTGGCGTCCTGGTACTGCTGG-3’ PM-113


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Which primers are included in which kit?

LEXSYcon kit with pLEXSY-sat

− Insert sequencing forward primer A992 − Insert sequencing reverse primer A264 − F4 reverse primer A1045 − sat forward primer F2999 − ssu forward primer F3001 − ssu reverse primer F3002

LEXSYcon kit with pLEXSY -hyg

− Insert sequencing forward primer A992 − Insert sequencing reverse primer A264 − F4 reverse primer A1045 − hyg forward primer A3804 − ssu forward primer F3001 − ssu reverse primer F3002

LEXSYcon kit with pLEXSY -ble

− Insert sequencing forward primer A992 − Insert sequencing reverse primer A264 − F4 reverse primer A1045 − ble forward primer A708 − ssu forward primer F3001 − ssu reverse primer F3002

LEXSYcon kit with pLEXSY -neo

− Insert sequencing forward primer A992 − Insert sequencing reverse primer A264 − F4 reverse primer A1045 − neo forward primer A1432 − ssu forward primer F3001 − ssu reverse primer F3002


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8.2. Preparation of LEXSY BHI agar plates for clonal selection • For 4 plates prepare 50 ml medium and bring to 37°C

Component Storage Amount / 100 ml

2x LEXSY BHI medium room temperature 35 ml inactivated Fetal Calf Serum (FCS)* -20°C 10 ml 1M HEPES, pH 7.4 4°C 4 ml Pen-Strep -20°C 1 ml Hemin (0,25% in 50% Triethanolamine) 4°C 0.2 ml

Selective antibiotic, if necessary

*Inactivated for 20 min at 56°C (or 1h at 52°C)

• Autoclave or melt (microwave) 50 ml 2% BACTO-Agar (DIFCO) and keep at 55°C

• Pour the medium into the warm agar, mix gently to avoid air bubbles, and distribute 25 ml per plate with serological pipette (air bubbles may be removed with the pipette).

• Dry the plates after solidifying for 10 min open under the laminar flow.

• Use the freshly prepared plates immediately, at least on the same day.

For your convenience, a LEXSY Plating Kit (Cat.-No. ML-451) containing everything for the preparation of 40 LEXSY BHI agar plates is available.


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8.3. Alternative electroporation: High-Voltage protocol for transfection of LEXSY • Grow L. tarentolae culture in LEXSY BHI medium to OD 1.0 (5x107 cells/ml).

• Take 1 ml of the culture and pellet the cells for 5 min at 2000g and 20°C

• Resuspend the cells in 0.5 ml LEXSY BHI medium

• Place this 0.5 ml culture into electroporation cuvette d=4 mm on wet ice

• Keep on ice 10 min

• Add 10 µg DNA in water or 10 mM Tris pH 8

• Pulse 2 times at 1500 V, 25 µF (10 sec between pulses)

• Put cuvettes back on ice EXACTLY 10 min

• Transfer to 10 ml LEXSY BHI kept at 26 °C

• Incubate o/n at 26°C

After 16-24h add antibiotic to culture for selection in suspension culture and/or spin 2 ml of culture 5 min/2000g, resuspend to 50 – 100 µl and plate carefully on LEXSY Broth BHI agar + selection antibiotic. Try to spread completely within shortest possible time to avoid stressing the cells.


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8.4. How to grow a Leishmania culture

For maintaining LEXSY strains for transfection and analysis, static suspension cultures should be grown in 10 ml LEXSY BHI with Hemin in ventilated cell culture flasks. Don´t use agitated cultures for strain maintenance since cells will age much faster.

For the vitality of LEXSY cultures it is important to support continuous growth by regularly transferring the strain at OD600nm in the range 2 - 3 (mid-late growth phase, 1 – 2 x108 cells/ml) into fresh medium at 1:10 to 1:50 dilutions. Use 1:10 dilutions during the week and 1:50 dilutions for maintaining the culture over the weekend, if appropriate. A 1:10 inoculated static suspension culture in ventilated cell culture flasks is ready for the next dilution usually after 3 days, a 1:50 inoculated culture after 5 days. To slow down growth rates you may position the TC flasks standing upright after initial growth for 3 days (lowering aeration), if longer intervals between passages are required. Don’t cultivate Leishmania longer than 7 days in the same medium when using 1:50 dilutions.

(1) The growth medium should always be fresh. To reach optimum growth and vitality the completed medium should be used within 2 weeks. The separate stock solutions of the medium components can be stored for a longer time (Hemin stock up to 1 year at 4°C, LEXSY antibiotics stocks up to 12 months at 4°C). During selection, add the antibiotic immediately before inoculation. Don’t use media, where the antibiotic was added a week before.

(2) Hemin, essential in the growth medium, is light-sensitive Leishmania must be cultivated in the dark. Also, the medium completed with Hemin must be stored in the dark (4°C).

For back-ups and in case of longer absence from the lab, prepare glycerol stocks of the LEXSY host and your cell lines as described (see section 3).

Glycerol stocks are best prepared from mid growth phase cultures @ 5x107 – 1x108 cells/ml; OD600nm 1.5 – 2.0. Avoid to prepare stocks from cultures not dense enough or from old, stationary phase cultures; ref. to (5). To reactivate, thaw the stocks slowly on ice and inoculate them directly into prewarmed (26°C) growth medium (with selective antibiotic, if applicable) in ventilated cell culture flasks. Use the entire content of the vial for 10 ml medium and do not refreeze glycerol stocks. Usually, you see motile cells immediately after inoculation. After 2 - 3 days the cultures from the first inoculation must be diluted 1:5 to 1:10 into fresh medium. Don’t keep the first inoculation culture from glycerol stocks longer than 4 days before passaging.

Do not use the first inoculation culture from glycerol stocks immediately for transfection. Passage the culture several times before electroporation, as described in section 5.

Always control appearance and motility of cells by microscopy. Cells of mid growth phase cultures are of drop-like shape, approx. 15 x 5 µm in size with one flagellum at the flat end, and motile. These cells are most efficient for transfection, plating on solid media and preparation of glycerol stocks. Mid growth phase cultures always contain subpopulations of non- or less motile cells and of cells of different shape. Don’t hesitate to transfect, plate or preserve a culture with drop like cells containing such subpopulations. Cells of older cultures get longer and thinner (needle-like shape) and remain motile. Enhanced motility may result from nutrient deprivation or other limitations and must not necessarily be a sign of mid growth culture stage. Also, bacterial, fungal or other contaminations may be identified by microscopy.

Keep patient, esp. if you are used to working with bacteria. Leishmania cells are protozoans with regular doubling times of 7 h in static suspension cultures and 4 - 5 h in agitated cultures. They need time to grow or to adapt to new conditions and sometimes they seem to be a bit inactive. Continuous inoculations into fresh medium, regular resuspension of sedimented cells in static suspension cultures in ventilated TC flasks (6), a dark place and some rest - and they will recover faster than you think.


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If you - despite following these instructions - eventually encounter growth problems with the host strain, sediment cells 5 min at 2000g, resuspend pellet carefully in fresh growth medium and continue incubation in ventilated cell culture flasks. This approach was very helpful in rescuing cultures.

Don’t centrifuge Leishmania cultures at high speed > 3000g and don’t resuspend cell pellets by rigorous vortexing. The cells are sensitive to these procedures and may lyse. Centrifugation at 2000g is sufficient for sedimentation and makes careful and quick resuspension of cell pellets easier. If required, prolong centrifugation time at 2000g rather than using higher speed.

Nobody is perfect. Please, don’t hesitate to contact the Jena Bioscience LEXSY team if you encounter problems with cultivation or for further questions.

Documents

LEXSYcon Expression Kit• The LEXSinduce kit (EGE-1200) contains one of the pLEXSY_I vectors (EGE-120 and EGE 121) for tetracycline-inducible bacteriophage-T7-based expression in