Expression of Human Proteins at the Southeast Collaboratory for Structural Genomics

Overview

Journal J Struct Funct Genomics

Specialty Genetics

Date 2004 Jul 21

PMID 15263854

Citations 10

Authors

Michael R Mayer

Tamara A Dailey

Clay M Baucom

Jill L Supernak

Michael C Grady

Harris E Hawk

Harry A Dailey

Affiliations

Soon will be listed here.

Abstract

The human protein production group at the Southeast Collaboratory for Structural Genomics is charged with producing human proteins for both X-ray crystallography and NMR structural studies. Eukaryotic, and human proteins in particular, are notoriously difficult to express in bacterial systems. For various reasons, T7-based expression often results in protein expressed in an insoluble form. Overcoming this requires either introduction of a step to screen expression conditions or inclusion of a troublesome refolding step during purification. Our laboratory uses a trc-based expression vector that addresses many of the difficulties of the commonly used T7-based expression systems. Proteins expressed under the trc promoter, a weak promoter compared to the strong T7 promoter, are produced in a soluble form and include necessary cofactors. The details of this system will be discussed.

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References

Dailey H, Sellers V, Dailey T . Mammalian ferrochelatase. Expression and characterization of normal and two human protoporphyric ferrochelatases. J Biol Chem. 1994; 269(1):390-5. View

Dailey T, Dailey H . Identification of [2Fe-2S] clusters in microbial ferrochelatases. J Bacteriol. 2002; 184(9):2460-4. PMC: 134991. DOI: 10.1128/JB.184.9.2460-2464.2002. View

Makrides S . Components of vectors for gene transfer and expression in mammalian cells. Protein Expr Purif. 1999; 17(2):183-202. DOI: 10.1006/prep.1999.1137. View

Connelly J, de Leau E, Okely E, Leach D . Overexpression, purification, and characterization of the SbcCD protein from Escherichia coli. J Biol Chem. 1997; 272(32):19819-26. DOI: 10.1074/jbc.272.32.19819. View

Edwards A, Arrowsmith C, Christendat D, Dharamsi A, Friesen J, Greenblatt J . Protein production: feeding the crystallographers and NMR spectroscopists. Nat Struct Biol. 2000; 7 Suppl:970-2. DOI: 10.1038/80751. View

Zhang Z, Song L, Fang M, Wang F, He D, Zhao R . Production of soluble and functional engineered antibodies in Escherichia coli improved by FkpA. Biotechniques. 2003; 35(5):1032-8, 1041-2. DOI: 10.2144/03355rr03. View

Dailey T, Dailey H . Human protoporphyrinogen oxidase: expression, purification, and characterization of the cloned enzyme. Protein Sci. 1996; 5(1):98-105. PMC: 2143237. DOI: 10.1002/pro.5560050112. View

Dailey T, Dailey H . Identification of an FAD superfamily containing protoporphyrinogen oxidases, monoamine oxidases, and phytoene desaturase. Expression and characterization of phytoene desaturase of Myxococcus xanthus. J Biol Chem. 1998; 273(22):13658-62. DOI: 10.1074/jbc.273.22.13658. View

Stevens R, Wilson I . Tech.Sight. Industrializing structural biology. Science. 2001; 293(5529):519-20. DOI: 10.1126/science.293.5529.519. View

10.

Richarme G, Caldas T . Chaperone properties of the bacterial periplasmic substrate-binding proteins. J Biol Chem. 1997; 272(25):15607-12. DOI: 10.1074/jbc.272.25.15607. View

11.

Bach H, Mazor Y, Shaky S, Berdichevsky Y, Gutnick D, Benhar I . Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies. J Mol Biol. 2001; 312(1):79-93. DOI: 10.1006/jmbi.2001.4914. View

12.

Tsumoto K, Ejima D, Kumagai I, Arakawa T . Practical considerations in refolding proteins from inclusion bodies. Protein Expr Purif. 2003; 28(1):1-8. DOI: 10.1016/s1046-5928(02)00641-1. View

13.

LaVallie E, McCoy J . Gene fusion expression systems in Escherichia coli. Curr Opin Biotechnol. 1995; 6(5):501-6. DOI: 10.1016/0958-1669(95)80083-2. View

14.

Hannig G, Makrides S . Strategies for optimizing heterologous protein expression in Escherichia coli. Trends Biotechnol. 1998; 16(2):54-60. DOI: 10.1016/s0167-7799(97)01155-4. View

15.

Sachdev D, Chirgwin J . Solubility of proteins isolated from inclusion bodies is enhanced by fusion to maltose-binding protein or thioredoxin. Protein Expr Purif. 1998; 12(1):122-32. DOI: 10.1006/prep.1997.0826. View

16.

Brosius J . Toxicity of an overproduced foreign gene product in Escherichia coli and its use in plasmid vectors for the selection of transcription terminators. Gene. 1984; 27(2):161-72. DOI: 10.1016/0378-1119(84)90137-9. View

17.

Stevens R . Design of high-throughput methods of protein production for structural biology. Structure. 2000; 8(9):R177-85. DOI: 10.1016/s0969-2126(00)00193-3. View

18.

Jeong K, Lee S . Secretory production of human granulocyte colony-stimulating factor in Escherichia coli. Protein Expr Purif. 2001; 23(2):311-8. DOI: 10.1006/prep.2001.1508. View

19.

Studier F, Rosenberg A, Dunn J, Dubendorff J . Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990; 185:60-89. DOI: 10.1016/0076-6879(90)85008-c. View

20.

Medlock A, Dailey H . Examination of the activity of carboxyl-terminal chimeric constructs of human and yeast ferrochelatases. Biochemistry. 2000; 39(25):7461-7. DOI: 10.1021/bi000134p. View