Allan Leck Jensen

Tools for the production and purification of full-length, N- or C-terminal 32P-labeled protein, applied to HIV-1 Gag and Rev.

We have constructed two new vectors for the production of foreign proteins in Escherichia coli. The vectors, pGEX-GTH and pET-HTG, produce protein fused to glutathione S-transferase (GST) at the N- and C-termini, respectively, allowing one-step purification on glutathione-Sepharose. Furthermore, they carry the recognition sequence (RRASV) for the catalytic subunit of cAMP-dependent heart muscle kinase (HMK) at the terminus distal to the GST tag, enabling specific 32P labeling in vitro. By positioning the GST and HMK sequences at opposite ends of the introduced gene, only full-length fusion protein becomes radiolabeled after purification. Avoiding the labeling of shorter fusion protein species, often observed in bacterial expression of foreign genes, is particularly important for a number of different purposes, including protein mobility shift analysis and protein footprinting technology.

Probing the structure of HIV-1 Rev by protein footprinting of multiple monoclonal antibody-binding sites

Human immunodeficiency virus type 1 (HIV‐1) Rev is a small RNA‐binding protein which is essential for viral replication. To investigate the structure of Rev we have mapped the binding sites of a panel of monoclonal antibodies (mAb) by protein footprinting and identified a mAb protecting amino acids within both the N‐ and C‐terminal parts of Rev. Our mapping results support a previously proposed structure (Auer et al., Biochemistry, 33 (1994) 2988–2996) predicting that a helix–loop–helix motif in Rev brings the termini of the protein into proximity. Furthermore, we demonstrate that the binding sites mapped by protein footprinting are in agreement with conventional epitope mapping results and that this technique provides an advantageous strategy for mapping discontinuous sites.