Eukaryotic expression library construction and screening experiments

Summary

Similar to Scheme 1, Scheme 2 describes the method of cDNA library construction and screening on eukaryotic expression vectors, and the process is divided into the following two stages: 1. construction of cDNA libraries on eukaryotic expression vectors; 2. screening of cDNA libraries constructed on eukaryotic expression vectors. This experiment was obtained from Molecular Cloning Laboratory Guide (3rd edition), author: Huang Peitang.

Operation method

Eukaryotic expression library construction and screening experiments

Materials and Instruments

Host cells Plasmids or λ phage expression vectors Packaged extracts Electro-transformed susceptible E. coli
Restriction endonuclease buffer Restriction endonuclease mRNA cDNA libraries enriched by poly(A)+ Terrific spheroplasts with appropriate antibiotics Terrific broth medium with appropriate antibiotics
cDNA Synthesis Kit

Move

Phase I: Construction of cDNA libraries on eukaryotic expression vectors

Material

Buffers and Solutions

10X Restriction Endonuclease Buffer

Enzymes & Buffers

Restriction Endonuclease
See step 3

Nucleic acids and oligonucleotides

Enrichment of mRNA by poly(A ⁾⁺
The mRNA should be extracted from cells or tissues expressing the target protein with self-activation and purified by two rounds of oligo(dT)-cellulose affinity chromatography (see Chapter 7, Scheme 3). The method for identifying tissues or cell lines with high expression of the target protein should be the same as the screening method used in the expression clones. Knowing the size of the target mRNA is useful for expression cloning, and the mRNA can be fractionated hierarchically, and the translational products of the different sized fractions can then be assayed for the biological activity of the target protein to determine whether this cross-graded mRMA fraction can be injected into oocytes to assay for the activity of target proteins in the eggs, or the mRNA fraction can be transfected into cultured mammalian cells (see Chapter 16).

Specialized Equipment

cDNA synthesis kit
These kits are available from a variety of manufacturers and typically include all of the reagents needed to prepare cDNA libraries and to ligate them to λ phage or plasmid vectors (see information block "Commercial cDNA Synthesis and Library Construction Kits").

Additional Reagents

The reagents required for Step 1 of this protocol are listed in Protocol 1, Stages 1-4.
The reagents required for Step 2 of this protocol are listed in Protocol 1, Phase 5.
The reagents required for steps 4 and 5 of this program are listed in Program 1, Phase 6.
Step 8 of this program may require reagents listed in Program 26 of Chapter 1.

Vectors and Strains

Electrotransformed susceptible E. coli
If a plasmid expression vector is chosen, prepare (see Chapter 1, Scheme 26) or purchase electro-transformed susceptible E. coli as host cells for the cDNA library. Transformation efficiencies should be at least ^5x108 clones/ul of plasmid DNA, and it is preferable to use only the same batch of prepared sensory bacteria for expression cloning and screening.

Packaging extracts
If a λ phage vector is chosen, purchase (or prepare) a high titer packaging extract for packaging the recombinant cDNA in λ viral particles (see "In Vitro Packaging" in the Information section). The titer of the packaging extract should be at least ¹⁰⁹ pfu/ug of viral DNA.

Plasmid or λ Phage Expression Vector
The choice of vector depends on the host system used for expression. If the host is Xenopus oocytes, expression vectors should be selected from plasmid vectors (e.g., expression vectors λZAP Express, λExCell, and pSPORT) in which the λ phage cultivar has an initiator derived from Sp6, T3, or T7 phage in the vicinity of the polyclonal site. If mammalian cells are used as hosts, plasmid vectors with strong promoters (e.g., human cytomegalovirus early transient promoter) and strong transcriptional termination sequences (e.g., the 3' end of the human growth hormone gene or the SV40 late terminator sequence) are selected. Such plasmids include the pCMV family of plasmids and plasmid pcDNA4 (see Table 11-5 and Appendix 3).

Methods

1. Prepare a flat-ended double-stranded cDNA using a commercial kit or according to Protocol 1 (Stages 1-4) and attach a suitable junction or articulator to the end.

2. Separate the double-stranded cDNAs by size gradation using gel filtration chromatography, as described in Scheme 1, Phase 5.
If the target mRNA size is known, collect the fraction of column chromatography product that is between the target mRNA size = 1kb. If the size is unknown, collect the column chromatography products in the following sizes: 500~1500bp,1500~3000bp and >300bp.

3. Double digest 10~25ug plasmids or λ phage expression vectors with corresponding sticky ends at the 5' and 3' ends of the junction-articulator region on the cDNA.
Important: Ensure that the digest is complete. Make sequential rather than simultaneous double cleavages; purify the DNA between cleavages by phenol/chloroform extraction combined with ethanol precipitation, and use gel electrophoresis whenever possible to check for completeness of cleavage.
To ensure complete digestion of the expression vector before cloning, some researchers insert a 200-300bp stuffer sequence between the multiple cloning sites SatⅠ and NatⅠ (or other combinations of double digestion sites). In this way, the digestion efficiency can be monitored at any time, and in some cases, the double digestion effect of the vector can be enhanced, because the two restriction sites in the polyclonal site region are usually very close to each other, and separating them by 200~300bp can eliminate the end inhibition of the digestion caused by this phenomenon.

4. Perform ligation pre-tests with different ratios of cDNA to vector (phage arm or plasmid DNA) molecules. See the Introduction to Stage 6 in Scheme 1 of this chapter for details on optimizing the ligation reaction (also see Step 1 of Stage 6).
If the CDNA has been divided into different sized fractions by hierarchical separation (see Note on Stage 2), each fraction must be tested separately for ligation efficiency.

5. Take a small portion of each ligation reaction product and package it in λ phage particles. Determine the titer of infected particles produced by each packaging reaction. For details, see the column "Lambda Phage Plating of E. coli Strains" and Steps 2-6 of Scheme 1, Phase 6. Alternatively, take a small portion of each ligation reaction product and transform E. coli by electroporation (see Scheme 26 in Chapter 1).

6. Determine whether the cDNAs on the six recombinant λ phage or plasmid vectors are of the right size, determine the optimal ratio of ligation reaction cDNAs to vector DNA that produces the maximum number of recombinant clones, and calculate the abundance of the cDNA libraries constructed according to the parameter settings.

7. Ligate as many cDNAs as possible to λ phage or plasmid DNA according to the ratio of cDNA inserts to vector molecules in the optimal ligation reaction.
It is common to set up many small ligation systems rather than one large reaction system.

8. Prepare and analyze the recombinants using one of the methods described below:
If λ phage vectors are used: package the ligation product DNA into λ phage particles according to the instructions provided by the supplier of the packaging extracts, determine the titer of the viral reservoir, and store it at 4°C.
If using a plasmid vector: Electrotransform E. coli with a small portion of the ligation reaction product to detect the number of possible recombinants generated by the ligation reaction (see Scheme 26 in Chapter 1).

9. Screening of the eukaryotic expression library should be performed as soon as possible.

Stage 2: Screening of cDNA libraries constructed on eukaryotic expression vectors

MATERIALS

Buffers and solutions
For storage solutions, see Appendix 1 for buffer and reagent components, dilute the storage solution in the appropriate proportions.

SM

Enzyme and buffer

Restriction endonucleases
Restriction endonucleic acids required for a specific program. See steps 1 and 3.

Nucleotides and Oligonucleotides

cDNA libraries, prepared as described in Stage 1 of this program.

Controls for transfection/injection experiments
It is difficult to overestimate the value of a cDNA that is set as a positive control in an expression clone. If possible, select a cDNA that encodes a protein whose biological activity is similar to that of the target protein to be cloned. For example, if you want to clone a cDNA for a new ^K+ channel protein, use a cDNA for a previously cloned ^K+ channel protein as a positive control. The control cDNA is used to determine the appropriate sublibrary capacity and whether the transformation/infection assay parameters are set appropriately. The positive control cDNA must be transferred into an appropriate E. coli strain (e.g., using a plasmid expression vector) or packaged into a λ phage particle before proceeding to Step 1. When using Xenopus oocytes for transfection/injection experiments, two negative controls must be introduced: the empty vector plasmid and the transcription reaction system components without template DNA. Also in step 1, phage particles packed with a bacteriophage expression empty vector or E. coli that have been transfected with the empty expression vector plasmid are mandatory.

Culture medium

Terrific spheroplasts with appropriate antibiotics
Terrific broth medium (or other rich medium) with appropriate antibiotics

Additional reagents

Steps 1 and 2 of this protocol may require commercial plasmid extraction kits (see Protocol 9 in Chapter 1).
Steps 1 and 3 of this protocol may require reagents from one of the transfection protocols in Chapter 16.
Reagents that may be required for Steps 1 and 3 of this protocol are listed in Chapter 9, Scheme 6.
Steps 1 and 3 of this protocol may require reagents necessary for the injection of mRNA into Xenopus oocytes, see Spector et al.
Reagents that may be required for Step 1 of this protocol are listed in Chapter 2, Schemes 1 or 5 and Schemes 23 or 24.
Step 1 of this protocol requires the reagents necessary to detect the biological activity of the protein encoded by the positive control cDNA.
Reagents that may be required for Step 2 of this protocol are listed in Chapter 1, Scheme 26 and Chapter 2, Scheme 1 and Scheme 23 or 24.
Step 4 of this protocol requires the reagents necessary to detect the biological activity of the target protein.
Reagents required for Step 6 of this protocol are listed in Chapters 7, 12, 15, and 16.

Cells and Tissues

Host Cells
Oocytes are obtained from female South African Xenopus laevis, which are available from a number of biological supply organizations (e.g., Carolina Biological Supply or Kons Scientific). The ability of the oocyte population to express the injected mRNA varies seasonally and is influenced by the age and physiological status of the donor animal. Difficulties with mRNA expression can sometimes be resolved by changing the source of supply of animals. Methods of oocyte isolation are described in Colman (1984), Spector et al. (1998a) and Julius et al. Monkey-derived COS cells or derived cell lines of the 293 human embryonic kidney cell line have been selected for use as host cells for transient expression in mammals because of their high transfection efficiency during transient transfection (Gluzman1981;Gorman1990 ). However, many other cell lines (e.g., CHO and NIH-3T3) have also been successfully used for this purpose (Naglich et al. 1992; Bates et al, 1993; Young et al. 1993).
Detection of the biological activity of target proteins in uninjected oocytes or untransformed transfected cells prior to expression cloning should ensure that the target protein activity in the expression host used is at a barely detectable level.

Methods.

1. Set up a series of pre-tests to optimize the transfection and expression system used to screen the library for target dDNA clones. It is preferable to optimize experiments using a cDNA that has been cloned earlier and for which a reliable method for detecting the biological activity of the protein encoded by this cDNA is already available.

If using eukaryotic plasmid expression vectors and cultured cell hosts

a. Pick a single colony of E. coli containing the positive control cDNA plasmid into 10 ml of enriched medium (e.g., Terrific Broth Medium with 1 selective antibiotic) containing 10, 100, 1000, 10,000, or 100,000 clones derived from the transformation of E. coli with the empty vector. shake the culture overnight at 37°C until saturated.

b. Purify the plasmid DNA using a commercial kit to a purity required for efficient transfection of cultured mammalian cells (see Table 1-6 in Scheme 9, Chapter 1).

c. Transfect cultured eukaryotic cells with multiple plasmid samples prepared by one or more of the methods described in Chapter 16 and assay for biological activity of the protein encoded by the positive control cDNA.
For cDNA library screening, use the transfection method that produces the greatest signal-to-noise ratio with an acceptably low background.

If a phage RNA polymerase (e.g., T3, T7, or SP6) is used to transcribe the cDNA on the plasmid expression vector and inject the product mRNA into Xenopus oocytes, it is recommended to use a phage RNA polymerase (e.g., T3, T7, or SP6).

a. Follow steps a and b above.

b. Linearize the collected and purified plasmid DNA using the rare cleavage site introduced at the 3' end of the cDNA during library construction, and then use it as a template for in vitro transcription to mRNA (see Scheme 6 in Chapter 9).

c. Inject the mRNA into Xenopus oocytes to test the biological activity of the protein encoded by the positive control cDNA, or transfect the mRNA into other appropriate cell lines.

If a λ phage vector containing a phage-encoded RNA polymerase promoter is used as an expression vector, the mRNA can be used as an expression vector.

a. Prepare a series of phage suspensions containing different ratios of empty λ phage vectors to recombinant λ phage vectors containing control cDNA (10:1, 100:1, 1000:1, etc.). Infect a suitable strain of E. coli with a large number of phage particles so that the bacterial moss is almost lysed into pieces or infected bacteria grown in liquid medium are completely lysed (see Scheme 1 or 5 in Chapter 2).

b. Collect prepared λ-phage DNA from plates (see Chapter 2, Scheme 23) or from liquid medium (see Chapter 2, Scheme 24).

c. Linearize the λ phage DNA using the rare cleavage site introduced at the 3' end of the CDNA during library construction and transcribe the cDNA to mRNA in vitro as described in Chapter 9, Scheme 6.

d. Inject the mRNA into Xenopus oocytes and test the biological activity of the protein encoded by the positive control cDNA.

2. Using the results obtained in Step 1 as a general guide, divide the cDNA library into appropriately sized screening sub-libraries, and use them to transform or transfect E. coli. Prepare plasmid or λ phage DNA for target cDNA screening.

If the expression library is constructed on a plasmid vector and the library is grown in solid medium, the cDNA library can be used as a guide.

a. Take a sufficient number of fractions (e.g., 50-100 fractions, each sufficient to produce approximately 1000 recombinants) from the ligation reaction mixture (Stage 1, Step 8) and individually and independently electrotransform E. coli. Spread all of the products from each fraction onto 1 Terrific agar plate containing the appropriate antibiotic and incubate overnight at 37°C.
The expected number of clones should be grown on each plate. For example, if the screened library should have a capacity of 50,000 clones, the linkage mixture used for electrotransformation should be sufficient to produce 50 plates with approximately 1,000 clones per plate. Transformation cultures can be grown in liquid medium as in step b before spreading the plates.

b. Estimate the number of clones on each plate. Scrape the clones from each plate into 5 ml of enrichment medium (e.g., Terrific broth medium with selective antibiotics). Culture to saturation and purify plasmid DNA for screening.

If expression libraries are constructed in plasmid vectors and libraries are grown in liquid medium

The advantage of this procedure is that it saves time and effort by eliminating the plate laying step. At the same time, the possibility of losing some independent clones due to poor growth on agar plates or mechanical damage is reduced.

a. Take a sufficient number of small portions (e.g., 50-100 portions, each sufficient to produce approximately 1000 recombinants) of the ligation reaction mixture (Stage 1, Step 8) and individually electrotransform E. coli. The number of recombinant clones in each culture should be the expected number.

b. Immediately after electrotransformation, add lml of antibiotic-free rich medium (e.g. SOC or Terrific broth medium) to each culture and incubate at 37°C with very slow shaking for 1 h. The short incubation period will allow expression of the antibiotic resistance gene carried by the plasmid and accumulation to a level that is sufficient to protect the transformed bacteria.

c. Inoculate a portion of the bacteria with 5 ml of culture medium. The plasmid DNA is purified for screening after the culture is saturated.

If the expression library is constructed in λ phage vector and 10,000 to 100,000 cDNA clones need to be screened, the plasmid DNA should be purified.

a. Prepare a semi-dense recombinant phage spot library by infecting a bacterial plate with an appropriate volume of the packaging mixture. Alternatively, completely lysed E. coli grown in liquid medium with a sufficient amount of the packaging mixture.

b. Isolate phage DNA by plate lysis or liquid culture (see Chapter 2, Scheme 23 or 24).

If the expression library is constructed in a λ phage vector and no more than 10,000 cDNA clones are to be screened, the expression library should be constructed in the λ phage vector and no more than 10,000 clones should be screened.

a. Infect bacterial plates with the same number of recombinant phages to be screened at a density of 1000 pfu per 100 mm plate.

b. After phage plaque formation, add 2~3 ml of SM to each plate to prepare low-titer plate lysate and calculate the titer.

c. Infect liquid bacterial cultures with low-titer plate lysates to prepare high-titer phage reservoirs.

d. Purify phage DNA by liquid culture (see Chapter 2, Scheme 24).

3. Transfer the purified clones to an appropriate background system to analyze their expression
If the clone is purified plasmid DNA: Transfect cultured mammalian cells with the plasmid DNA or use it as an in vitro transcription template to prepare mKNA with phage RNA polymerase and inject it into Xenopus oocytes.
If the clone is purified λ-phage DNA: Linearize the λ-phage DNA, use it as the template for in vitro transcription to synthesize mRNA and inject it into Xenopus oocytes.
A typical transfection experiment uses 50~100 petri dishes of cultured cells to obtain 50,000^100,000 cDNA clones (1,000 individual clones per petri dish), which are screened for expression of the target bioactivity. In contrast, a typical sublibrary of mRNA injected into 5 Xenopus oocytes containing 1000 individual clones per sublibrary would require 250~500 oocytes for screening of 50,000~100,000 cDNAs for biological activity.

4. Detect the biological activity of the protein encoded by the target cDNA.

5. After identifying the positive sub-library, divide it into smaller and smaller libraries and repeat the test until the target cDNA clone is isolated and identified. The simplest way to accomplish this is to use recombinant plasmids or λ phage DNA purification products from positive sublibraries as follows:

a. Divide the positive (primary) sublibrary of plasmid or phage DNA into small portions, each of which can yield about one-tenth as many recombinant transforming clones or phage spots as the primary sublibrary.
A primary sublibrary containing 10,000 individual clones should be divided into approximately 30 portions, with each portion yielding about 1,000 clones or phage spots.

b. Repeat steps 3 and 4 above. The primary positive sublibrary should be introduced as a positive control in the repeat screening.

c. Repeat the sorting steps until a single recombinant is obtained that expresses the target biological activity. It is important to ensure that the phage or plasmid DNA used for the final screen is derived only from a single phage spot or bacteriophage clone.

6. Identify the selected cDNA monoclones encoding the target bioactivity using DNA sequencing (Chapter 12), expression (Chapter 15 or 16), and RNA hybridization (Chapter 7) methods.
If the recombinants do not contain all of the coding region of the cDNA, the initial cDNA selection can be used as a probe to screen additional recombinants from the cDNA library using high-rigor hybridization.

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