Protein interaction module-assisted function X (PIMAX) approach to producing challenging proteins including hyperphosphorylated tau and active CDK5/p25 kinase complex

Abstract

Many biomedically critical proteins are underrepresented in proteomics and biochemical studies because of the difficulty of their production in Escherichia coli. These proteins might possess posttranslational modifications vital to their functions, tend to misfold and be partitioned into bacterial inclusion bodies, or act only in a stoichiometric dimeric complex. Successful production of these proteins requires efficient interaction between these proteins and a specific "facilitator," such as a protein-modifying enzyme, a molecular chaperone, or a natural physical partner within the dimeric complex. Here we report the design and application of a protein interaction module-assisted function X (PIMAX) system that effectively overcomes these hurdles. By fusing two proteins of interest to a pair of well-studied protein-protein interaction modules, we were able to potentiate the association of these two proteins, resulting in successful production of an enzymatically active cyclin-dependent kinase complex and hyperphosphorylated tau protein, which is intimately linked to Alzheimer disease. Furthermore, using tau isoforms quantitatively phosphorylated by GSK-3β and CDK5 kinases via PIMAX, we demonstrated the hyperphosphorylation-stimulated tau oligomerization in vitro, paving the way for new Alzheimer disease drug discoveries. Vectors for PIMAX can be easily modified to meet the needs of different applications. This approach thus provides a convenient and modular suite with broad implications for proteomics and biomedical research.

Fig. 1.

A, design of PIMAX and its three applications described in this work. PIMAX stands for protein interaction module–assisted function X. To use PIMAX, the protein of interest (A) and its “facilitator” (B) are each fused to the Fos or Jun leucine zipper domain (black and gray “saw blades”). The highly efficient and specific dimerization of Fos and Jun helps bring about more effective interaction between A and B. Depending on the molecular and functional relationship, different products are obtained that support a variety of downstream applications. PPI, protein–protein interaction. In the application of PIMAX-Cat (catalysis), B is a modifying enzyme for A. The Fos–Jun association helps B modify A at a higher efficiency (A*). In the chaperone-assisted folding (CAF) mode, the folding and solubility of A are enhanced by a molecular chaperone. Finally, in the heterodimer co-purification (CoP) application of PIMAX, A and B are two subunits of a heterodimer. With the assistance of Fos and Jun, A–B association is maintained throughout the purification process, resulting in the purification of an active A–B complex for downstream applications. Note that although Fos and Jun leucine zippers form a parallel dimer, this scheme does not suggest that proteins A and B only associate with each other in the manner shown here. Because of the small size of Fos and Jun here, as well as the presence of additional amino acids between the PIMs and proteins A and B (see the supplemental material for the complete sequences of PIMAX vectors), a certain degree of flexibility may exist that allows A and B to establish a productive spatial orientation. B, basic features of PIMAX core vectors, including the Fos and Jun leucine zipper domains, hexahistidine (His) tag, protease sites, and ligation-independent cloning boxes to be cut by Asc I or NotI before the insertion of DNA fragments for proteins A and B. Additional application-specific elements such as the FLAG tag on the second recombinant gene are detailed in the corresponding sections and figures. C, recombinant proteins produced by different PIMAX extensions in this work.

Fig. 2.

Hyperphosphorylation of tau by GSK-3β via PIMAX-Cat. A, bacterial whole cell lysates (lanes 1–6) and eluates from Ni beads (lanes 7–10) were resolved via SDS-PAGE and stained with Coomassie Blue. Fos and Jun leucine zipper domains were too small to see with this gel. B, phosphatase treatment eradicated the GSK-3β-dependent tau mobility shift. Unphosphorylated and GSK-3β-co-expressed tau were isolated via Ni affinity purification. The imidazole eluates were then subjected to calf intestinal phosphatase treatment. C, inclusion of both Fos and Jun leucine zippers increased tau phosphorylation by GSK-3β. Jun–tau was co-expressed with GSK-3β with or without the Fos domain. Total tau was purified through boiling followed by perchloric acid extraction, which together removed most bacterial proteins, and analyzed via Coomassie Blue staining (CBR, upper panel) and immunobloting with an anti-tau antibody (lower panel).

Fig. 3.

Phosphorylation enhances tau fibrillization in vitro. A, final products of tau and hyperphosphorylated tau (p-tau) produced via PIMAX-Cat. These proteins were purified in their native form, that is, through Ni chromatography, TEV digestion, and gel filtration chromatography. Both leucine zipper domains and the His tag were removed (see “Experimental Procedures” for details). Lane 2, unphosphorylated tau; lanes 3 and 4, tau hyperphosphorylated by CDK5 and GSK-3β, respectively. B, immunoblotting analysis of tau and p-tau. Comparable amounts of the three tau species were loaded onto a nitrocellulose membrane with a slot blotter. Western blotting using antibodies against general tau or one of the indicated phospho-epitopes was conducted. The epitope for each antibody is listed above each strip. C, in vitro tau aggregation assays comparing the kinetics of tau and p-tau fibrillization in the absence or presence of heparin (hep.). The time-dependent increase in tau fibrillization was quantified by the fluorescence of thioflavin T (ThT) upon binding to amyloid protein complexes.

Fig. 4.

PIMAX-Cat to produce acetylated and phosphorylated tumor suppressor p53. A, relevant features of PIMAX-Cat plasmids for p53 PTMs. B, Aurora A kinase caused p53 to shift gel mobility when both Fos and Jun leucine zippers were present. Whole cell lysates were resolved via SDS-PAGE and stained with Coomassie Blue. C, D, Aurora A–mediated p53 phosphorylation also diminished immunoreactivity with the Ab-1 monoclonal antibody. The epitope for Ab-1 detection is shown, with the potential Aurora A phosphorylation site Ser215 highlighted. C shows the results of Western blots that probed bacterial lysates with two p53 antibodies. In D, Aurora A–co-expressed p53 was purified by Ni affinity beads, λ phosphatase (PPase) treatment, and then Western blotting. Phosphatase treatment changed p53 mobility and restored reactivity with the Ab-1 antibody. The lower band detectable by both p53 antibodies seen in C is likely an artifact of bacterial whole cell extracts, as well as overexposure of the Western blotting assays. E, two different acetylated p53 species generated by co-expressing p53 with acetyltransferases Gcn5 and p300 in the context of PIMAX-Cat. Bacterial lysates were analyzed via Western blotting using the indicated antibodies.

Fig. 5.

p53 solubility was significantly improved by PIMAX-CAF that exploited the molecular chaperone activity of TcFKBP18. A, relevant features of PIMAX-CAF p53 plasmid. B, Coomassie Blue–stained protein gel resolving soluble (S) and insoluble (P) fractions of bacterial lysates. The position of the full-length p53 fused to the Jun leucine zipper is marked with an asterisk, and the Fos–FKBP fusion protein is labeled with a pound sign.

Fig. 6.

Production of an enzymatically active cyclin-dependent kinase complex, CDK5/p25, with PIMAX-CoP. A, key features of PIMAX-CoP plasmid. See the text for an explanation of some of these elements. Note that TcFKBP18 drastically increased the yield of the complex. The following example thus is a combination of CAF and CoP. B, C, purification of CDK5/p25 complex through sequential His tag (B) and FLAG tag (C) affinity chromatography. CDK5 and p25 were liberated from the anti-FLAG matrix by means of either glycine elution (lane 6) or TEV digestion (lane 7). D, recombinant tau phosphorylation by CDK5/p25 complex produced by PIMAX. The mobility of tau before and after reaction with the kinase complex was examined via SDS-PAGE and Coomassie Blue staining.