Structure-function studies of an engineered scaffold protein derived from stefin A. I: Development of the SQM variant.
Non-antibody scaffold proteins are used for a range of applications, especially the assessment of protein-protein interactions within human cells. The search for a versatile, robust and biologically neutral scaffold previously led us to design STM (stefin A triple mutant), a scaffold derived from the intracellular protease inhibitor stefin A. Here, we describe five new STM-based scaffold proteins that contain modifications designed to further improve the versatility of our scaffold. In a step-by-step approach, we introduced restriction sites in the STM open reading frame that generated new peptide insertion sites in loop 1, loop 2 and the N-terminus of the scaffold protein. A second restriction site in 'loop 2' allows substitution of the native loop 2 sequence with alternative oligopeptides. None of the amino acid changes interfered significantly with the folding of the STM variants as assessed by circular dichroism spectroscopy. Of the five scaffold variants tested, one (stefin A quadruple mutant, SQM) was chosen as a versatile, stable scaffold. The insertion of epitope tags at varying positions showed that inserts into loop 1, attempted here for the first time, were generally well tolerated. However, N-terminal insertions of epitope tags in SQM had a detrimental effect on protein expression.
Hoffmann
Toni
T
Section of Experimental Therapeutics, Leeds Institute of Molecular Medicine, St James's University Hospital, Beckett St, Leeds LS97TF, UK. t.hoffmann@leeds.ac.uk
Stadler
Lukas Kurt Josef
LK
Busby
Michael
M
Song
Qifeng
Q
Buxton
Anthony T
AT
Wagner
Simon D
SD
Davis
Jason J
JJ
Ko Ferrigno
Paul
P
D019943:Amino Acid Substitution; D002942:Circular Dichroism; D055312:Cystatin A / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli; D047468:Immunoprecipitation; D046228:Microarray Analysis; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D013329:Structure-Activity Relationship
D055312:Cystatin A