Structure-Expression Relationship of Tumor Necrosis Factor Receptor Mutants That Increase Expression
The extracellular domain of the p55 TNF receptor (TNFrED) is an important therapeutic protein for targeting tumor necrosis factor-α (TNF-α). The expression level of the TNFrED is low for bioproduction, which is presumably associated with the complication of pairing 24 cysteine residues to form corre...
Saved in:
Published in | The Journal of biological chemistry Vol. 278; no. 31; pp. 28961 - 28967 |
---|---|
Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
United States
Elsevier Inc
01.08.2003
American Society for Biochemistry and Molecular Biology |
Subjects | |
Online Access | Get full text |
Cover
Loading…
Summary: | The extracellular domain of the p55 TNF receptor (TNFrED) is an important therapeutic protein for targeting tumor necrosis factor-α (TNF-α). The expression level of the TNFrED is low for bioproduction, which is presumably associated with the complication of pairing 24 cysteine residues to form correct disulfide bonds. Here we report the application of the yeast display method to study expression of TNFrED, a multimeric receptor. Randomly mutated libraries of TNFrED were screened, and two mutants were identified that express several-fold higher protein levels compared with the wild type while still retaining normal binding affinity for TNF-α. The substituted residues responsible for the higher protein expression in both mutants were identified as proline, and both proline residues are adjacent to cysteine residues involved in disulfide bonds. Analysis of the mutant residues revealed that the improved level of expression is due to conformational restriction of the substituted residues to that of the folded state seen in the crystal structures of TNFrED thereby forcing the neighboring cysteine residues into the correct orientation for proper disulfide bond formation. |
---|---|
Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M212019200 |