Structure-Expression Relationship of Tumor Necrosis Factor Receptor Mutants That Increase Expression

Rene L Schweickhardt,Xuliang Jiang,Louise M Garone,William H Brondyk

doi:10.1074/jbc.m212019200

Rene L Schweickhardt, Xuliang Jiang + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m212019200

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Abstract

The extracellular domain of the p55 TNF receptor (TNFrED) is an important therapeutic protein for targeting tumor necrosis factor-alpha (TNF-alpha). 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-alpha. 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.

Highlights

The ability to obtain a high level of expression of secreted recombinant proteins in yeast and mammalian cells is often found to be protein-dependent
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
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

Summary

EXPERIMENTAL PROCEDURES

Construction of Yeast and Mammalian Expression Plasmids—The TNFrED-agglutinin fusion was constructed by linking the signal sequence from the invertase gene to the human TNFrED sequence encoding residues 12–172, which was fused to the C-terminal portion of the ␣-agglutinin gene encoding residues 330 – 650. Purified plasmid DNA was re-transformed into BJ2168 yeast cells, and individual clones were re-analyzed using methods described above. TNF-␣ Binding Assay—Yeast expressing either the wild-type or mutant TNFrED-agglutinin fusion or yeast containing the control vector pYES2 were resuspended in PBS/bovine serum albumin (10 mg/ml) at a concentration of 1 ϫ 108 cells/ml. In each well of a Durapore 96multiwell plate (Millipore Corp.), 50 ␮l of the cell suspension were incubated with 50 ␮l of PBS/bovine serum albumin (10 mg/ml) containing various concentrations of 125I-TNF-␣ (Amersham Biosciences) for 2 h at room temperature. Surface Plasmon Resonance Analysis of TNFrED Mutants—Surfaces displaying polyclonal goat anti-human TNFrED were constructed by binding biotinylated antibody (BAF225 from R&D Systems) to a Sensor SA chip (P/N BR-1000-32, BIAcore Inc.), which was conjugated with streptavidin. Where R is the gas constant, Az is the accessible conformational area in the Ramachandran conformational plot for residue Z, and AX is that for residue X

RESULTS

TNFrED and mutants of TNFrED

TABLE II Binding parameters fit to SPR data

DISCUSSION