Ijh

Amylose resin

MalE FUSION PROTEIN BINDS MALTOSE AND IS RELEASED FROM COLUMN

MalE FUSION PROTEIN BINDS MALTOSE AND IS RELEASED FROM COLUMN

Treat with protease Xa

Target protein

FIGURE 26.11 Maltose-Binding Protein Fusion Vector

In order to manufacture an MBP-tagged protein, the target gene is cloned into the pMAL vector. The vector has a polylinker downstream from the malE gene for inserting the gene of interest. For correct translation, the target gene must be cloned in frame with the malE gene. The entire region between the strong tac promoter and the strong terminator produces a fusion protein consisting of MBP (MalE protein) linked to the target protein via a protease cleavage site. The lacZa sequence allows for blue/white color screening to detect DNA insertion (see Ch. 22).

used. Bacteria defective in several protease genes are useful, even though not all

E. coli protease genes can be inactivated since some are essential for survival.

Self Cleavable Intein Tags

A recent improvement in tagging systems eliminates the protease cleavage and purification step. Instead of a protease recognition site, the fusion protein cleaves itself after purification of the target protein. This approach is based on the properties of inteins, self-splicing intervening sequences that are found in proteins (see Ch. 12). The advantages are a reduction in the number of steps required for purification and the avoidance of expensive proteases that may sometimes cleave the protein of interest at other sites.

The Intein Mediated Purification with Affinity Chitin-binding Tag (IMPACTTM) system from New England Biolabs depends on the self-splicing of an intein originally from the VMA1 gene of Saccharomyces cerevisiae. This intein has been modified to undergo self-cleavage only at its N-terminus. This is triggered at low temperatures by thiol reagents such as dithiothreitol (DTT). At the C-terminal end of the intein is a small chitin binding domain (CBD) from the carboxy terminus of the chitinase A1 gene of Bacillus circulans. [Chitin is a structural polymer of N-acetyl glucosamine. It is found in most fungi and is the major structural component of the exoskeletons of insects.]

The gene encoding the target protein is inserted into a multiple cloning site (MCS) upstream of the gene encoding the intein plus chitin-binding domain (CBD). The fusion protein is purified by binding to a chitin column. While the fusion protein is still attached to the column, intein self-cleavage is induced by incubation at 4°C with DTT. The target protein is released while the intein plus the chitin binding domain remain bound to the column (Fig. 26.12).

Selection by Phage Display

Most procedures in molecular biology deal with either genetic information (i.e., DNA or RNA) or gene products (i.e., proteins). Display protocols are designed to provide both the gene and its encoded protein together. The most common of these is intein Self-splicing intervening sequence that is found in a protein

Tags based on inteins are convenient because they can be designed to cleave themselves off when no longer required.

Phage display allows us to find a gene by identifying the protein it encodes.

FIGURE 26.12 Intein Mediated Purification System

Inteins that can self-cleave at the amino-terminus allow specific proteins to be purified and cleaved from a fusion protein in one step. First, the target gene is cloned upstream of the intein sequence and a chitin binding domain. The fusion gene is transcribed and translated in bacteria. The bacteria are lysed and release a mixture of proteins that are passed through a chitin column. The proteins with the chitin binding domain bind to the chitin and the remaining proteins pass through. Adding DTT and incubating at 4°C activates the intein to cleave itself from the target protein, which is therefore released from the column.

tac promoter

Chitin binding domain tac promoter

Chitin binding domain

Transcription and translation

Protein

Protein

POUR INTO chiTIN COLUMN

POUR INTO chiTIN COLUMN

Induce self-cleavage

Induce self-cleavage

Target protein released

Chitin

Target protein released

Chitin

The proteins to be screened are fused to virus proteins so that they appear on the outside surface of the virus particle.

the phage display technique. Here a full-length protein or a shorter peptide is fused to a coat protein of a bacteriophage so as to be displayed on the outer surface of the virus particle. Meanwhile, the DNA encoding the fusion protein is carried inside the bacteriophage (Fig. 26.13).

Filamentous phage M13 is most popular choice for phage display but Lambda, T7 and T4 are also used. M13 is preferred since it is non-lytic and does not destroy the host bacteria during phage production. Instead, phage particles are secreted through the bacterial cell envelope. The absence of cell debris simplifies purification of the phage. Three M13 structural proteins have been used as expression platforms for the proteins of interest with gene III protein most popular. There are about 2,500 copies of the major coat protein (gene VIII protein) on the phage surface but only five copies of the minor coat protein (gene III protein). Having fewer copies of the displayed peptide on the surface of the phage avoids artifacts due to simultaneous binding of multiple polypeptides. Since the N-terminus of the M13 coat proteins is external and the C-terminal region interacts with the DNA inside the phage particle, the displayed peptide must be attached to the N-terminus of gene III.

The DNA encoding the protein or peptide is fused to the gene for the phage coat protein by a PCR-based technique. The linear PCR product is amplified and circularized to give a viral genome that is transformed into E. coli cells. The phages that are phage display Fusion of a protein or peptide to the coat protein of a bacteriophage whose genome also carries the cloned gene encoding the protein. The protein is displayed on the outside of the virus particle and the corresponding gene is carried on the inside

FIGURE 26.13 Principle of Phage Display

In order to display a peptide on the surface of a bacteriophage, the DNA sequence encoding the peptide must be fused to the gene for a bacteriophage coat protein. In this example, the chosen coat protein is encoded by gene III of phage M13. Here the N-terminal portion will be on the outside of the phage particle whereas the C-terminus will be on the inside. Therefore the peptide must be fused in frame at the N-terminus to be displayed on the outside of the phage.

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