title: An efficient system for small protein expression and refolding.
abstract: The low expression yield and poor refolding efficiency of small recombinant proteins expressed in Escherichia coli have continued to hinder the large-scale purification of such proteins for structural and biological investigations. A system based on a small fusion partner, the B1 domain of Streptococcal protein G (GB1), was utilized to overcome this problem. We have tested this system on a small cysteine-rich toxin, mutant myotoxin alpha (MyoP20G). The highly expressed fusion protein was refolded using an unfolding/refolding protocol. Due to the small size of GB1, we were able to monitor the unfolding/refolding status by heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The final product yielded well-resolved NMR spectra, with a topology corresponding to the natural product. We conclude that GB1 not only increases the expression level but also enhances the refolding of small proteins.
authors.affiliation: Structural Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA. chengy@mskcc.org
authors.forename: Yuan
authors.initials: Y
authors.lastname: Cheng
authors.forename: Dinshaw J
authors.initials: DJ
authors.lastname: Patel
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003435:Crotalid Venoms / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005240:Feasibility Studies; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D018014:Gene Transfer Techniques; D024861:Genetic Enhancement / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D003435:Crotalid Venoms; C034284:IgG Fc-binding protein, Streptococcus; D011993:Recombinant Fusion Proteins; C018932:myotoxin A