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https://www.um.edu.mt/library/oar/handle/123456789/120639
Title: | GST fusion protein expression vector for in-frame cloning and site directed mutagenesis |
Authors: | Hunter, Therese Hunter, Gary J. |
Keywords: | Molecular cloning Deoxyribonucleases Escherichia coli -- Genetics Gene expression Genetic vectors Glutathione transferase Mutagenesis Plasmids -- Genetics Superoxide dismutase |
Issue Date: | 1998 |
Publisher: | Future Science Ltd |
Citation: | Hunter, T., & Hunter, G. J. (1998). GST fusion protein expression vector for in-frame cloning and site-directed mutagenesis. Biotechniques, 24(2), 194-196. |
Abstract: | A novel glutathione S-transferase (GST) fusion protein expression vector, pGHX(-), was constructed to include a bacteriophage f1 origin of replication and a unique SalI restriction site. The former enables the packaging of the vector into filamentous bacteriophage for the isolation of single-stranded (ss)DNA, while the latter was engineered to form part of the DNA that encodes the protease recognition site for factor Xa, downstream of the gene encoding GST (Figure 1). These modifications allow both site-directed mutagenesis and the subsequent expression of the encoded GST fusion protein using a single plasmid. This obviates the need for subcloning mutated foreign genes from a single-stranded vector into an expression plasmid and therefore avoids many time-consuming protocols such as DNA digestion, fragment isolation, ligation, transformation and screening. Importantly, when the SalI site is used for blunt-end cloning, the amino acid sequence of the subsequently purified protein corresponds precisely and only to the encoding DNA of the cloned insert. No spurious N-terminal amino acids due to unavoidable cloning methodologies will be present in the purified protein product. This is particularly relevant to studies involving protein-protein interactions of purified binding domains and to studies of organelle protein targeting that involve N-terminal signal sequences. |
URI: | https://www.um.edu.mt/library/oar/handle/123456789/120639 |
Appears in Collections: | Scholarly Works - FacM&SPB |
Files in This Item:
File | Description | Size | Format | |
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GST_fusion_protein_expression_vector_for_in_frame_cloning_and_site_directed_mutagenesis.pdf | 59.71 kB | Adobe PDF | View/Open |
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