Short, hydrophobic, alanine-based proteins based on the basic region/leucine zipper protein motif: overcoming inclusion body formation and protein aggregation during overexpression, purification, and renaturation.
GCN4 is a yeast transcriptional regulatory protein; its DNA-binding domain is a basic region/leucine zipper (bZIP) structure that comprises a dimer of alpha-helices capable of high-affinity, sequence-specific recognition of the DNA major groove. We are exploiting what nature has evolved by manipulating the bZIP motif as a molecular recognition scaffold; thus we reduced the elegantly minimal bZIP to an even more simplified structure by substitution with alanine residues-hence, a generic, Ala-based, helical scaffold. These Ala-based mutants are unusual proteins for expression as they are short ( approximately 100 amino acids) and hydrophobic (Ala-mutated basic regions, leucine-zipper dimerization domains). Hydrophobicity posed a major problem throughout the expression, isolation, and purification stages; inclusion body formation and protein aggregation were significant hurdles throughout protein production. We describe measures that solved these problems, including use of high concentrations of denaturant in all steps of protein isolation and purification and use of temperature-dependent renaturing techniques to obtain folded, functional protein. Despite these difficulties, we ultimately retrieved 5-10 mg/L of broth of active, correctly folded protein after the complete purification procedure. Homogeneity of the proteins was established by chromatography, electrophoresis, and mass spectrometry. Furthermore, characterization by circular dichroism and DNase footprinting analysis demonstrates that these alanine-based mutants retain the structure and function of the native GCN4 DNA-binding domain. Remarkably, the most heavily mutated protein, containing 24 alanines of 27 total amino acids in the DNA-binding basic region, still binds the AP-1 site, the target of native GCN4.
Lajmi
A R
AR
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania, 15260, USA.
Wallace
T R
TR
Shin
J A
JA
D000409:Alanine / Q000737:chemistry*; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002479:Inclusion Bodies / Q000378:metabolism*; D016350:Leucine Zippers; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020673:Protein Renaturation; D029701:Saccharomyces cerevisiae Proteins
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D011494:Protein Kinases; D000409:Alanine