PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

Eduardo Hilario; Li Fan; Leonard  J. Mueller; Michael  F. Dunn

doi:10.3791/61839

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PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

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生化学

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JoVE Journal 生化学

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

4,062 Views

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09:31 min

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September 26, 2020

DOI:

10.3791/61839-v

DOI:

10.3791/61839-v

•

09:31 min

September 26, 2020

•

5 Views

Eduardo Hilario¹, Li Fan², Leonard J. Mueller¹, Michael F. Dunn²

¹化学科,University of California-Riverside, ²Department of Biochemistry,University of California-Riverside

概要

This article presents a series of consecutive methods for the expression and purification of Salmonella typhimurium tryptophan synthase comp this protocol a rapid system to purify the protein complex in a day. Covered methods are site-directed mutagenesis, protein expression in Escherichia coli, affinity chromatography, gel filtration chromatography, and crystallization.

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PCR Mutagenesis Cloning Expression Protein Purification Tryptophan Synthase Salmonella Typhimurium Ammonium Sulfate Fractionation Size Exclusion Chromatography SDS-PAGE Protein Concentration Crystallization

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PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

概要

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