Refolding of serine proteinases

  title={Refolding of serine proteinases},
  author={A. Light and C. Duda and T. W. Odorzynski and W. G. Moore},
  journal={Journal of Cellular Biochemistry},
Bovine trypsinogen and chymotrypsinogen were successfully refolded as the mixed disulfide of glutathione using cysteine as the disulfide interchange catalyst. The native structures were regenerated with yields of 40%–50% at pH 8.6 and 4 °C, and the half‐time for the refolding was approximately 60–75 min. We then refolded threonine‐neochymotrypsinogen, which is a two‐chain structure held together by disulfide bonds and produced on cleavage of Tyr 146‐Thr 147 in native chymotrypsinogen [Duda CT… Expand
Bacterial expression and refolding of human trypsinogen.
This work describes the high level expression of human trypsinogen 1 in E. coli using the T7 expression system and the utility for the detachment of mammalian cells in culture was proven. Expand
Isolation, renaturation, and formation of disulfide bonds of eukaryotic proteins expressed in Escherichia coli as inclusion bodies
Which methods originally developed for studying the folding mechanism of naturally occurring proteins have been successfully adapted for reactivation of recombinant eukaryotic proteins are shown. Expand
Expression of human cationic trypsinogen with an authentic N terminus using intein-mediated splicing in aminopeptidase P deficient Escherichia coli.
An expression system that produces recombinant human cationic trypsinogen with a native, intact N terminus is described, using intein-mediated protein splicing and an aminopeptidase P (pepP) deficient E. coli strain. Expand
Renaturation of recombinant proteins produced as inclusion bodies.
  • B. Fischer
  • Chemistry, Medicine
  • Biotechnology advances
  • 1994
Renatured and native disulphide bond formation are accomplished by (a) either air oxidation, (b) glutathione reoxidation starting from reduced material, or (c) disulPHide interchange starting from mixed disULphides containing peptides. Expand
Trypsin Mutants for Structure-Based Drug Design: Expression, Refolding and Crystallisation
The use of surrogate proteins for studying protein ligand interactions and the selection of bovine trypsin as a scaffold to reconstruct the ligand binding site of factor Xa are investigated. Expand
Solubility as a Function of Protein Structure and Solvent Components
  • C. Schein
  • Chemistry, Medicine
  • Bio/Technology
  • 1990
Special attention is given to areas where solubility limitations pose major problems, as in the preparation of highly concentrated solutions of recombinant proteins for structural determination with NMR and X-ray crystallography, refolding of inclusion body proteins, studies of membrane protein dynamics, and the formulation of proteins for pharmaceutical use. Expand


Refolding of the mixed disulfide of bovine trypsinogen and glutathione.
The mixed disulfide of bovine trypsinogen and glutathione refolded with high yields at protein concentrations of 20 microgram/ml or less, at 4-25 degrees C, pH 8.0 to 8.7, behaved as native trypsInogen as judged by gel exclusion chromatography, isoelectric focusing, and activation with bovines enterokinase or tryps inactivation. Expand
Refolding of reduced, denatured trypsinogen and trypsin immobilized on Agarose beads.
The reoxidation of fully reduced and denatured bovine trypsinogen and the regeneration of the native structure can be accomplished if the protein is initially attached to Agarose beads, and the rate-limiting step in the refolding of trypsInogen was disulfide interchange. Expand
Refolding of bovine threonine-neochymotrypsinogen.
  • C. Duda, A. Light
  • Medicine, Chemistry
  • The Journal of biological chemistry
  • 1982
These studies show that two separate fully reduced polypeptide chains were capable of refolding, associating with one another, and regenerating a native structure with full biological activity. Expand
Studies on the reduction and re-formation of protein disulfide bonds.
This simple and elegant experiment was designed to determine what would happen when reduced, unfolded, and enzymatically inactive ribonuclease was allowed simply to refold in the presence of only neutral aqueous buffer and air. Expand
Crystal structure of bovine trypsinogen at 1-8 A resolution. II. Crystallographic refinement, refined crystal structure and comparison with bovine trypsin.
Three chain segments forming the specificity pocket in active trypsin and consisting of residues Gly142 to Pro152, GlyA184 to Gly193 and Gly216 to Asn223 show no significant continuous electron density in the final Fourier map. Expand
Crystal structure of bovine trypsinogen at 1-8 A resolution. I. Data collection, application of patterson search techniques and preliminary structural interpretation.
A preliminary difference Fourier map showed several interesting details of the overall chain folding in bovine trypsinogen, which indicates that the N terminus may be only weakly fixed to the rest of the molecule or may even float freely in solution. Expand
The anatomy and taxonomy of protein structure.
This chapter investigates the anatomy and taxonomy of protein structures, based on the results of three-dimensional X-ray crystallography of globular proteins. Expand
Principles of Protein Structure
1 Amino Acids.- 1.1 The 20 Standard Amino Acids.- 1.2 Why Were Just These Amino Acids Selected?.- 1.3 Colinear Relation Between Nucleic Acids and Polypeptides.- 1.4 Side Chain Properties.- 1.5Expand
Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4
Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four majorExpand
Principles that govern the folding of protein chains.
In his Nobel Lecture, Anfinsen provided a sketch of the rich history of research that provided the foundation for his work on protein folding and the "Thermodynamic Hypothesis," and outlined potential avenues of current and future scientific exploration. Expand