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Fermenter production of an artificial fab fragment, rationally designed for the antigen cystatin, and its optimized crystallization through constant domain shuffling.
Proteins. 1995 Dec; 23(4):561-5.P

Abstract

The synthetic antibody model "M41" was rationally designed with a binding site complementary to chicken egg white cystatin as the prescribed antigen. In order to permit comparison between the computer model and an experimental three-dimensional structure of the artificial protein, its X-ray crystallographic analysis was pursued. For this purpose, M41 was expressed as a recombinant Fab fragment in E. coli by medium cell density fermentation employing the tightly regulated tetracycline promoter. The Fab fragment was efficiently purified via a His-6 tail fused to its heavy chain and immobilized metal affinity chromatography. To raise the chances for the productive formation of crystal packing contacts, three versions of the Fab fragment were generated with differing constant domains. One of these, the variant with murine C kappa and CH1 gamma 1 domains, was successfully crystallized by microseeding in a sitting drop. The orthorhombic crystals exhibited symmetry of the space group P2(1)2(1)2(1) with unit cell dimensions a = 104.7 A, b = 113.9 A, c = 98.8 A and diffracted X-rays to a nominal resolution of 2.5 A.

Authors+Show Affiliations

Schiweck W
Abteilung Molekulare Membranbiology, Max-Planck-Institut für Biophysik, Frankfurt am Main, Germany.
Skerra A
No affiliation info available

MeSH

Amino Acid SequenceAnimalsChickensCloning, MolecularComputer SimulationCrystallizationCrystallography, X-RayCystatinsElectrophoresis, Polyacrylamide GelEscherichia coliFermentationImmunoglobulin Fab FragmentsMiceModels, MolecularMolecular Sequence DataMutagenesis, Site-DirectedPoint MutationProtein ConformationRecombinant Proteins

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

8749852

Citation

Schiweck, W, and A Skerra. "Fermenter Production of an Artificial Fab Fragment, Rationally Designed for the Antigen Cystatin, and Its Optimized Crystallization Through Constant Domain Shuffling." Proteins, vol. 23, no. 4, 1995, pp. 561-5.
Schiweck W, Skerra A. Fermenter production of an artificial fab fragment, rationally designed for the antigen cystatin, and its optimized crystallization through constant domain shuffling. Proteins. 1995;23(4):561-5.
Schiweck, W., & Skerra, A. (1995). Fermenter production of an artificial fab fragment, rationally designed for the antigen cystatin, and its optimized crystallization through constant domain shuffling. Proteins, 23(4), 561-5.
Schiweck W, Skerra A. Fermenter Production of an Artificial Fab Fragment, Rationally Designed for the Antigen Cystatin, and Its Optimized Crystallization Through Constant Domain Shuffling. Proteins. 1995;23(4):561-5. PubMed PMID: 8749852.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Fermenter production of an artificial fab fragment, rationally designed for the antigen cystatin, and its optimized crystallization through constant domain shuffling. AU - Schiweck,W, AU - Skerra,A, PY - 1995/12/1/pubmed PY - 1995/12/1/medline PY - 1995/12/1/entrez SP - 561 EP - 5 JF - Proteins JO - Proteins VL - 23 IS - 4 N2 - The synthetic antibody model "M41" was rationally designed with a binding site complementary to chicken egg white cystatin as the prescribed antigen. In order to permit comparison between the computer model and an experimental three-dimensional structure of the artificial protein, its X-ray crystallographic analysis was pursued. For this purpose, M41 was expressed as a recombinant Fab fragment in E. coli by medium cell density fermentation employing the tightly regulated tetracycline promoter. The Fab fragment was efficiently purified via a His-6 tail fused to its heavy chain and immobilized metal affinity chromatography. To raise the chances for the productive formation of crystal packing contacts, three versions of the Fab fragment were generated with differing constant domains. One of these, the variant with murine C kappa and CH1 gamma 1 domains, was successfully crystallized by microseeding in a sitting drop. The orthorhombic crystals exhibited symmetry of the space group P2(1)2(1)2(1) with unit cell dimensions a = 104.7 A, b = 113.9 A, c = 98.8 A and diffracted X-rays to a nominal resolution of 2.5 A. SN - 0887-3585 UR - https://www.unboundmedicine.com/medline/citation/8749852/Fermenter_production_of_an_artificial_fab_fragment_rationally_designed_for_the_antigen_cystatin_and_its_optimized_crystallization_through_constant_domain_shuffling_ L2 - https://doi.org/10.1002/prot.340230411 DB - PRIME DP - Unbound Medicine ER -