Recombinant DNA Technology

Recombinant DNA Technology - EUKARYOTIC EXPRESSION LIBRARIE

슬로싱킹 2024. 10. 27. 12:13

 

EUKARYOTIC EXPRESSION LIBRARIES

In expression libraries, the vector has sequences required for transcription and translation of the insert. This means that the insert DNA is expressed as RNA and then translated into a protein. An expression library, in essence, generates a protein from every cloned insert, whether it is a real gene or not.

 

When eukaryotic DNA is studied, expression libraries are constructed using complementary DNA (cDNA) to help ensure the insert is truly a gene. Eukaryotic DNA, especially in higher plants and animals, is largely noncoding, with coding regions spaced far apart. Even genes are interrupted with noncoding introns.

 

cDNA is a double-stranded DNA copy of mRNA. cDNA is made by reverse transcriptase, an enzyme first identified in retroviruses (see Chapter 1). It is used in eukaryotic research to eliminate the introns and generate a version of a gene consisting solely of an uninterrupted coding sequence.

 

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In contrast, bacteria have very little noncoding DNA, and their genes are not interrupted by introns; therefore, genomic DNA can be used directly in expression libraries. Eukaryotic DNA is first made into cDNA in order to construct an expression library (Fig. 3.21).

FIGURE 3.21 Making a cDNA Library from Eukaryotic mRNA First, eukaryotic cells are lysed and the mRNA is purified. Next, reverse transcriptase plus primers containing oligo(dT) stretches are added. The oligo(dT) hybridizes to the adenine in the mRNA poly(A) tail and acts as a primer for reverse transcriptase. This enzyme makes the complementary DNA strand, forming an mRNA/cDNA heteroduplex. The mRNA strand is digested with ribonuclease H, and DNA polymerase I is added to synthesize the opposite DNA strand, thus creating double-stranded cDNA. Next, S1 nuclease is added to trim off any single-stranded ends, and linkers are added to the ends of the dsDNA. The linkers have convenient restriction enzyme sites for cloning into an expression vector.

 

 

To make cDNA, the messenger RNA is isolated from the organism of interest by binding to a column containing poly(T) (i.e., a DNA strand consisting of repeated thymines). This isolates only mRNA because poly(T) anneals to the poly(A) tail of eukaryotic mRNA.

The mRNA is converted into cDNA using reverse transcriptase, which synthesizes a DNA complement to mRNA. An enzyme then removes the mRNA part of the mRNA/cDNA heteroduplex, and DNA polymerase makes the second strand of DNA (see Fig. 3.21). The final product is a double-stranded DNA copy of the mRNA sequence. The cDNA is then ligated into an expression vector with sequences that initiate transcription and translation of the insert. In some cases, the insert will have its own translation start site (e.g., a full-length cDNA). If the insert does not contain a translation start, then the reading frame becomes an issue. Because the genetic code is triplet, each insert can be translated in three different reading frames. A protein may be produced for all three reading frames, but only one frame will actually produce the correct protein. To ensure obtaining inserts with the correct reading frame, each cDNA is cloned in all three reading frames by using linkers with several different restriction sites. The number of transformants to screen for a protein of interest is therefore increased. The cloned genes are transformed into bacteria, which express the foreign DNA. The bacteria are grown on agar, and the colonies are then transferred to a nylon membrane and lysed. The proteins released are attached to the nylon membranes and are screened in various ways. Most often, an antibody to the protein of interest is used (see Chapter 6). This recognizes the protein and can be identified using a secondary antibody that is conjugated to a detection system. Usually, alkaline phosphatase is conjugated to the secondary antibody. The whole complex can be identified because alkaline phosphatase cleaves X-Phos, leaving a blue color where the bacterial colony expressed the right protein (Fig. 3.22).

FIGURE 3.22 Immunological Screening of an Expression Library Bacteria expressing foreign genes are grown on an agar plate, transferred to a membrane, and lysed. Released proteins are bound to the membrane. This figure shows only one attached protein, although in reality many different proteins are present. These include both expressed library clones and bacterial proteins. The membrane is incubated with a primary antibody that binds only the protein of interest. To detect this protein:antibody complex, a second antibody with a detection system such as alkaline phosphatase is added. The bacterial colony expressing the protein of interest will turn blue when X-Phos is added. This allows the vector with the correct insert to be isolated.

 

 

E. coli cannot perform most of the post-translational modifications that eukaryotic proteins often undergo. Therefore, the proteins are not always in their native form. Nonetheless, appropriate antibodies can detect most proteins of interest.

 

Complementary DNA or cDNA is constructed by isolating mRNA and making a DNA copy with reverse transcriptase. Expression libraries express the foreign DNA insert as a protein because expression vectors contain sequences for both transcription and translation. The protein of interest is identified by incubating the library with an antibody to the protein of interest.