Ribonuclease S‐peptide as a carrier in fusion proteins

Abstract

S-peptide (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and S-protein (residues are the enzymatically inactive products of the limited digestion of ribonuclease A by subtilisin. S-peptide binds S-protein with high affinity to form ribonuclease S, which has full enzymatic activity. Recombinant DNA technology was used to produce a fusion protein having three parts: carrier, spacer, and target. The two carriers used were the first 15 residues of S-peptide (S15) and a mutant S15 in which Asp 14 had been changed to Asn (D14N S15). The spacer consisted of three proline residues and a fourresidue sequence recognized by factor X, protease. The target was /3-galactosidase. The interaction between the S-peptide portion of the fusion protein and immobilized S-protein allowed for affinity purification of the fusion protein under denaturing (S15 as carrier) or nondenaturing (D14N S15 as carrier) conditions. A sensitive method was developed to detect the fusion protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by its ribonuclease activity following activation with S-protein. S-peptide has distinct advantages over existing carriers in fusion proteins in that it combines a small size (215 residues), a tunable affinity for ligand (Kd L lo-' M), and a high sensitivity of detection (~1 0 "~ mol in a gel).

Autoimmune-associated PTPN22 R620W Variation Reduces Phosphorylation of Lymphoid Phosphatase on an Inhibitory Tyrosine Residue

“…An S-tag (15 aa; see Ref. 19) was cloned for 3Ј and was in-frame with the HA tag in the pEF5 vector, thus generating the pEF5HA-S vector. LYP mutants were then subcloned into the BamHI site.…”

Assembly of Arenavirus Envelope Glycoprotein GPC in Detergent-Soluble Membrane Microdomains

“…The attenuated Candid#1 strain of JUNV (5,42) was provided by Robert Tesh (WHO Reference Center for Arboviruses) and was propagated in Vero cells. The Z protein cDNA from the Candid#1 virus (28) was kindly provided by Victor Romanowski (National University of La Plata, La Plata, Argentina) and was subsequently engineered to encode a C-terminal S peptide (Spep) tag (31,77). The GPC and Z proteins of MC2 and Candid#1 are essentially identical, each containing two conservative amino acid substitutions among 485 and 94 residues, respectively.…”

“…For double labeling of Z and GPC, GPC was again visualized with anti-G1 antibody and 12-nm-diameter gold particles, while Z was detected using biotinylated S protein (Novagen), which binds an Spep affinity tag (31) appended to the C terminus of Z, and streptavidin conjugated to 6-nm-diameter gold particles. Thin sections were scanned at low magnification to identify transfected cells that had positive immunoreactivity, and arbitrary regions of plasma membrane were imaged at high magnification (ϫ43,000).…”

“…We therefore wanted to investigate whether the distribution of GPC on the plasma membrane might be affected by coexpression of Z. In these studies, GPC was again visualized with an anti-G1 antibody labeled with 12-nm-diameter gold particles, and because no specific antibodies directed to Z are available, coexpression of the matrix protein was detected using an Spep affinity tag (31) appended to the C terminus of Z. This construct had been shown to support the budding of virus-like particles when coexpressed with GPC (J. York and S. S. Agnihothram, unpublished data), and analogously tagged Z proteins have been used successfully in reverse-genetic studies with LCMV (52).…”

Bitopic Membrane Topology of the Stable Signal Peptide in the Tripartite Junín Virus GP-C Envelope Glycoprotein Complex

“…A 15-amino-acid S-peptide (Spep) affinity tag (25) was introduced into the recombinant SSP to examine the localization of the N and C termini of SSP. We have previously shown that Spep could be appended to the C terminus of SSP without affecting the ability of the SSP subunit to trans-complement a G1-G2 precursor bearing the conventional signal peptide of CD4 (CD4sp-GPC) (49).…”