The domains of protein G from streptococcus which bind immunoglobulin G have been cloned and expressed in Escherichia coli (Fahnestock et al., 1986). Because protein G binds to several animal immunoglobulin G's, it has many immunochemical applications. This report describes process development for large-scale production of this recombinant protein G (also known as GammaBind G). In 200 l cultures of E. coli, this protein G variant was released from the cell into the culture medium by heating at 80 degrees C for 10 min. The concentration was monitored by either a competitive enzyme-linked immunoassay or a liquid chromatographic assay. Cross-flow microfiltration with 0.22 micron membrane was used to remove the cells. The protein G-rich permeate from the cross-flow microfilter was purified by affinity chromatography using a 5 l column of IgG-Sepharose 6 Fast Flow, which yielded 16-18 g of protein G per column cycle. The pools of purified protein G were concentrated and desalted using ultrafiltration. The salt-free protein G was then lyophilized as bulk product. The overall recovery through the entire process was 50-64%. The analysis of the final product included sodium dodecyl sulfate polyacrylamide gel electrophoresis, UV-visible spectrum, high performance gel filtration, endotoxin level and binding efficiency to human IgG Sepharose.

01 Oct 1991·BiochemistryQ3 · BIOLOGY

Conformational stability, folding, and ligand-binding affinity of single-chain Fv immunoglobulin fragments expressed in Escherichia coli

Q3 · BIOLOGY

Article

Author: Hardman, Karl D. ; Wood, Jay F. ; Dodd, Steven W. ; Bird, Robert E. ; Johnson, Syd ; Pantoliano, Michael W. ; Asel, Eric D.

A fluorescein-binding single-chain Fv (scFv) was chosen as a model for the study of the physicochemical parameters associated with synthetic IgG fragments. Three such scFv proteins were designed from the primary sequences of one anti-fluorescyl monoclonal antibody (Mab 4.4.20). These were constructed with varying-length interdomain peptide linkers of between 12 and 25 residues, expressed in Escherichia coli, and the protein folding, stability, and antigen-binding characteristics were assessed. Efficient renaturation could be accomplished in vitro to yield approximately 26 mg of active scFv/L of fermentation. Scatchard analysis for fluorescein ligand binding revealed that the scFv designs come within 2-fold of the Ka = 1.99 (+/- 0.18) x 10(9) observed for the parental 4.4.20 Fab and have identical stoichiometries (n approximately 0.99). Reversible solvent denaturation studies demonstrated that the unfolding/refolding equilibria for the scFv proteins can be fit to a simple two-state model and that two of the scFv designs were found to be slightly more stable than single IgG domains (VL and CL) when assessed in terms of the free energy of unfolding, delta Gon-u, or nearly identical to other multiple domain immunoglobulin proteins such as light chains and Fab's when relative transition midpoints, Cm, are compared. Linkers which conferred conformational flexibility beyond the minimally required length of 12 residues were found to have a stabilizing effect. By these criteria of ligand-binding function and protein stability, the scFv proteins were found to be bona fide minimal replicas of their parental IgG molecules.

09 Aug 1991·ScienceQ1 · CROSS-FIELD

A Novel, Highly Stable Fold of the Immunoglobulin Binding Domain of Streptococcal Protein G

Q1 · CROSS-FIELD

Article

Author: Achari, Aniruddha ; Filpula, David R. ; Clore, G. Marius ; Essig, Nina Z. ; Wingfield, Paul T. ; Whitlow, Marc ; Gronenborn, Angela M.

The high-resolution three-dimensional structure of a single immunoglobulin binding domain (B1, which comprises 56 residues including the NH 2 -terminal Met) of protein G from group G Streptococcus has been determined in solution by nuclear magnetic resonance spectroscopy on the basis of 1058 experimental restraints. The average atomic root-mean-square distribution about the mean coordinate positions is 0.27 angstrom (Å) for the backbone atoms, 0.65 Å for all atoms, and 0.39 Å for atoms excluding disordered surface side chains. The structure has no disulfide bridges and is composed of a four-stranded β sheet, on top of which lies a long helix. The central two strands (β1 and β4), comprising the NH2- and COOH-termini, are parallel, and the outer two strands (β2 and β3) are connected by the helix in a +3 x crossover. This novel topology (-1, +3 x , -1), coupled with an extensive hydrogen-bonding network and a tightly packed and buried hydrophobic core, is probably responsible for the extreme thermal stability of this small domain (reversible melting at 87°C).

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