Overproduction and Dissection of Proteins by the Expression-cassette Polymerase Chain Reaction

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 1990 Mar 1

PMID 2408046

Citations 25

Authors

K D MacFerrin

M P Terranova

S L Schreiber

G L Verdine

Affiliations

Soon will be listed here.

Abstract

We report an efficient, general approach for the construction of protein-overproducing strains of Escherichia coli. The method, expression-cassette polymerase chain reaction (ECPCR), allows the insertion of virtually any contiguous coding sequence between sequences that direct high-level protein biosynthesis in E. coli. The gene expression cassettes obtained by ECPCR are inserted into a regulated overexpression plasmid, and the resulting construct is used to transform E. coli. By effecting simultaneous 5' and 3' modification of a coding sequence, ECPCR permits the facile generation of mutant proteins having N- and/or C-terminal truncations. The method is a highly efficient way to dissect a multidomain protein into its component domains. The efficiency of the ECPCR approach is demonstrated in this study by construction of permuted overexpression vectors for the first two extracellular domains of the human CD4 protein.

Citing Articles

NMR spectroscopy reveals unexpected structural variation at the protein-protein interface in MHC class I molecules.

Beerbaum M, Ballaschk M, Erdmann N, Schnick C, Diehl A, Uchanska-Ziegler B J Biomol NMR. 2013; 57(2):167-78.

PMID: 24006098 DOI: 10.1007/s10858-013-9777-z.

Step-wise loss of bacterial flagellar torsion confers progressive phagocytic evasion.

Lovewell R, Collins R, Acker J, OToole G, Wargo M, Berwin B PLoS Pathog. 2011; 7(9):e1002253.

PMID: 21949654 PMC: 3174259. DOI: 10.1371/journal.ppat.1002253.

Pseudomonas aeruginosa evasion of phagocytosis is mediated by loss of swimming motility and is independent of flagellum expression.

Amiel E, Lovewell R, OToole G, Hogan D, Berwin B Infect Immun. 2010; 78(7):2937-45.

PMID: 20457788 PMC: 2897393. DOI: 10.1128/IAI.00144-10.

RcoM: a new single-component transcriptional regulator of CO metabolism in bacteria.

Kerby R, Youn H, Roberts G J Bacteriol. 2008; 190(9):3336-43.

PMID: 18326575 PMC: 2347395. DOI: 10.1128/JB.00033-08.

Origin of the 2-amino-2-deoxy-gluconate unit in Rhizobium leguminosarum lipid A. Expression cloning of the outer membrane oxidase LpxQ.

Que-Gewirth N, Karbarz M, Kalb S, Cotter R, Raetz C J Biol Chem. 2003; 278(14):12120-9.

PMID: 12531908 PMC: 2548327. DOI: 10.1074/jbc.M300379200.

References

Chaudhary V, Mizukami T, Fuerst T, Fitzgerald D, Moss B, Pastan I . Selective killing of HIV-infected cells by recombinant human CD4-Pseudomonas exotoxin hybrid protein. Nature. 1988; 335(6188):369-72. DOI: 10.1038/335369a0. View

Huston J, Levinson D, Tai M, Novotny J, Margolies M, Ridge R . Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci U S A. 1988; 85(16):5879-83. PMC: 281868. DOI: 10.1073/pnas.85.16.5879. View

Gold L . Posttranscriptional regulatory mechanisms in Escherichia coli. Annu Rev Biochem. 1988; 57:199-233. DOI: 10.1146/annurev.bi.57.070188.001215. View

Littman D, Maddon P, Axel R . Corrected CD4 sequence. Cell. 1988; 55(4):541. DOI: 10.1016/0092-8674(88)90211-5. View

Capon D, Chamow S, Mordenti J, Marsters S, Gregory T, Mitsuya H . Designing CD4 immunoadhesins for AIDS therapy. Nature. 1989; 337(6207):525-31. DOI: 10.1038/337525a0. View

White J, Littman D . Viral receptors of the immunoglobulin superfamily. Cell. 1989; 56(5):725-8. DOI: 10.1016/0092-8674(89)90674-0. View

Chao B, Costopoulos D, Curiel T, Bertonis J, Chisholm P, Williams C . A 113-amino acid fragment of CD4 produced in Escherichia coli blocks human immunodeficiency virus-induced cell fusion. J Biol Chem. 1989; 264(10):5812-7. View

Traunecker A, Schneider J, Kiefer H, Karjalainen K . Highly efficient neutralization of HIV with recombinant CD4-immunoglobulin molecules. Nature. 1989; 339(6219):68-70. DOI: 10.1038/339068a0. View

Edelman G . Topobiology. Sci Am. 1989; 260(5):76-82, 84-6, 88. DOI: 10.1038/scientificamerican0589-76. View

10.

Arthos J, Deen K, Chaikin M, Fornwald J, Sathe G, Sattentau Q . Identification of the residues in human CD4 critical for the binding of HIV. Cell. 1989; 57(3):469-81. DOI: 10.1016/0092-8674(89)90922-7. View

11.

Dahlberg A . The functional role of ribosomal RNA in protein synthesis. Cell. 1989; 57(4):525-9. DOI: 10.1016/0092-8674(89)90122-0. View

12.

Chaudhary V, Queen C, Junghans R, Waldmann T, Fitzgerald D, Pastan I . A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin. Nature. 1989; 339(6223):394-7. DOI: 10.1038/339394a0. View

13.

Reid K, Day A . Structure-function relationships of the complement components. Immunol Today. 1989; 10(6):177-80. DOI: 10.1016/0167-5699(89)90317-4. View

14.

Ward E, Gussow D, Griffiths A, Jones P, Winter G . Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature. 1989; 341(6242):544-6. DOI: 10.1038/341544a0. View

15.

Siekierka J, Hung S, Poe M, Lin C, Sigal N . A cytosolic binding protein for the immunosuppressant FK506 has peptidyl-prolyl isomerase activity but is distinct from cyclophilin. Nature. 1989; 341(6244):755-7. DOI: 10.1038/341755a0. View

16.

Harding M, Galat A, Uehling D, Schreiber S . A receptor for the immunosuppressant FK506 is a cis-trans peptidyl-prolyl isomerase. Nature. 1989; 341(6244):758-60. DOI: 10.1038/341758a0. View

17.

Lamarre D, Capon D, Karp D, Gregory T, Long E, Sekaly R . Class II MHC molecules and the HIV gp 120 envelope protein interact with functionally distinct regions of the CD4 molecule. EMBO J. 1989; 8(11):3271-7. PMC: 401455. DOI: 10.1002/j.1460-2075.1989.tb08487.x. View

18.

Huse W, Sastry L, Iverson S, Kang A, Alting-Mees M, Burton D . Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science. 1989; 246(4935):1275-81. DOI: 10.1126/science.2531466. View

19.

Tabor S, Richardson C . A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985; 82(4):1074-8. PMC: 397196. DOI: 10.1073/pnas.82.4.1074. View

20.

Laemmli U . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0. View