Cloning, expression, and purification of human cyclophilin in Escherichia coli and assessment of the catalytic role of cysteines by site-directed mutagenesis

Journal Article · Thu Mar 01 00:00:00 EST 1990 · Proceedings of the National Academy of Sciences of the United States of America; (USA)

DOI:https://doi.org/10.1073/pnas.87.6.2304· OSTI ID:6073593

Liu, J; Chen, C; Walsh, C T ^[1]; Albers, M W ^[2]; Schreiber, S L ^[3]

---

1. Harvard Medical School, Boston, MA (USA)
2. Harvard Medical School, Boston, MA (USA) Harvard Univ., Cambridge, MA (USA)
3. Harvard Univ., Cambridge, MA (USA)

The cDNA encoding human cyclophilin from the Jurkat T-cell lymphoma line has been cloned by the expression cassette polymerase chain reaction and sequenced, and an expression vector has been constructed under control of the tac promoter for efficient expression in Escherichia coli. Active cyclophilin is produced at up to 40% of soluble cell protein, facilitating a one-column purification to homogeneity. Wild-type cyclophilin was characterized for binding of the potent immunosuppressant agent cyclosporin A by tryptophan fluorescence enhancement and for inhibition of cyclophilin's peptidyl-proly cis-trans isomerase (rotamase) activity. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as the substrate, recombinant human cyclophilin has a high catalytic efficiency. To test the prior suggestion that a cysteine residue may be essential for catalysis and immunosuppressant binding, the four cysteines at positions 52, 62, 115, and 161 were mutated individually to alanine and the purified mutant proteins were shown to retain full affinity for cyclosporin A and equivalent catalytic efficiency as a rotamase. Clearly the cysteines play no essential role in catalysis or cyclosporin A binding. These results rule out the recently proposed mechanism. Involving the formation of tetrahedral hemithioorthoamide. Whereas mechanisms that embody other tetrahedral intermediates may be operative, an alternative mechanism is considered that involves distortion of bound substrate with a twisted (90{degree}) peptidyl-prolyl amide bond.

• OSTI ID: 6073593
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (USA), Vol. 87:6; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
MEMBRANE PROTEINS
BIOCHEMICAL REACTION KINETICS
RECOMBINANT DNA
DNA-CLONING
CYSTEINE
DNA POLYMERASES
DNA SEQUENCING
DOSE-RESPONSE RELATIONSHIPS
ELECTROPHORESIS
ESCHERICHIA COLI
ISOMERASES
LYMPHOMAS
MUTAGENESIS
PHOSPHORUS 32
PURIFICATION
TUMOR CELLS
AMINO ACIDS
ANIMAL CELLS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CLONING
DAYS LIVING RADIOISOTOPES
DISEASES
DNA
DNA HYBRIDIZATION
ENZYMES
HYBRIDIZATION
IMMUNE SYSTEM DISEASES
ISOTOPES
KINETICS
LIGHT NUCLEI
MICROORGANISMS
NEOPLASMS
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
STRUCTURAL CHEMICAL ANALYSIS
THIOLS
TRANSFERASES
550201* - Biochemistry- Tracer Techniques