{"doi":"10.1016/j.ab.2025.116014","title":"One-step purification of a bioactive PAK1-derived peptide","abstract":"The serine/threonine kinase PAK1 serves as a mediator of cytoskeletal reorganization and cancer-related signaling downstream of the small GTPases. Due to the challenges in purifying PAK1 complexes, a 46-residue peptide from PAK1, is widely used to study PAK1-Cdc42 signaling. Traditionally, this purification involved multi-step chromatography of recombinant GST-PBD46 complexes, yielding approximately 1 mg per 1.5 L culture. In this study, a 30 min heat treatment step after thrombin cleavage was used to precipitate GST while leaving pure PBD46 in solution. This step eliminated the need for further affinity and size- exclusion chromatography steps. This improved protocol produces proteins with a 6.5-fold higher yield, halves the purification processing time, and produces peptides with ≥95 % purity. Mass spectrometry, CD, fluorescence, and 1 H- 15 N HSQC NMR confirmed the heat-purified PBD46’s identity, structure, folding, and binding to Cdc42. The method also successfully separated other small peptides (e.g., ACK1) but not larger folded proteins. This rapid and scalable approach facilitates peptide production and biochemical studies without compromising the structural or functional integrity. We believe that the method described herein is applicable to other stable GST-fused recombinant proteins. • The PAK1’s p21-binding domain (PBD) is essential to binding Cdc42 and other Rac1 GTPases. A simple and more scalable method to isolate these peptides is needed to study these interactions. • In this study, we present an alternative to conventional purification of GST-bound peptides. A single 30-minute heating step is performed after thrombin cleavage to remove GST while PBD46 remains soluble. This eliminates an additional chromatography step. • The protocol presented increases protein yield while shortening purification time in comparison to standard, multi-column purification. • The desired peptide is highly pure and intact as indicated by LC-MS/MS and NMR. It also binds Cdc42 with nanomolar affinity as expected. • This method can be extended to other small, heat-stable peptides but does not work for larger (> 20kD) proteins.","journal":"Analytical Biochemistry","year":2025,"id":581876,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9588,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1493763,"name":"Emily P Esquivel","orcid":null,"position":1,"is_corresponding":false},{"id":1493764,"name":"Stacy R Hunter","orcid":null,"position":2,"is_corresponding":false},{"id":757498,"name":"Patience Okoto","orcid":"0000-0002-3828-6804","position":3,"is_corresponding":false},{"id":564983,"name":"Thallapuranam Krishnaswamy Suresh Kumar","orcid":"0000-0001-7262-1373","position":4,"is_corresponding":false},{"id":1493408,"name":"Paul D. Adams","orcid":"0000-0002-4823-1145","position":5,"is_corresponding":false},{"id":757497,"name":"Djamali Muhoza","orcid":"0000-0002-3480-335X","position":0,"is_corresponding":true}],"reference_count":17,"raw_metadata":null,"created_at":"2026-07-19T02:58:51.328454Z","pmid":"41265809","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}