Efficient high level expression of peptides and proteins as fusion proteins with the N-terminal domain of L9: application to the villin headpiece helical subdomain.
The efficient expression of small to midsize polypeptides and small marginally stable proteins can be difficult. A new protein fusion system is developed to allow the expression of peptides and small proteins. The polypeptide of interest is linked via a Factor Xa cleavage sequence to the C-terminus of the N-terminal domain of the ribosomal protein L9 (NTL9). NTL9 is a small (56 residue) basic protein. The C-terminus of the protein is part of an alpha-helix which extends away from the globular structure thus additional domains can be fused without altering the fold of NTL9. NTL9 expresses at high levels, is extremely soluble, and remains fully folded over a wide temperature and pH range. The protein has a high net positive charge, facilitating purification of fusion proteins by ion exchange chromatography. NTL9 fusions can also be easily purified by reverse phase HPLC. As a test case we demonstrate the high level expression of a small, 36 residue, three helix bundle, the villin headpiece subdomain. This protein is widely used as a model system for folding studies and the development of a simple expression system should facilitate experimental studies of the subdomain. The yield of purified fusion protein is 70 mg/L of culture and the yield of purified villin headpiece subdomain is 24 mg/L of culture. We also demonstrate the use of the fusion system to express a smaller marginally folded peptide fragment of the villin headpiece domain.
Bi
Yuan
Y
Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY 11794-3400, USA.
Tang
Yuefeng
Y
Raleigh
Daniel P
DP
Cho
Jae-Hyun
JH
D003001:Cloning, Molecular; D006801:Humans; D008840:Microfilament Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D008956:Models, Chemical; D008958:Models, Molecular; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D012269:Ribosomal Proteins / Q000096:biosynthesis / Q000235:genetics*
D008840:Microfilament Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C049650:ribosomal protein L9; C026615:villin