Signal peptide does not inhibit binding of biotin to streptavidin.
Three recombinant polypeptides of streptavidin: the full-length streptavidin with a signal peptide (rsavS), full-length streptavidin (rsavF) and core streptavidin (rsavC), were expressed in E. coli strain BL21 (DE3) and purified by Ni-NTA chromatography. Although all three recombinant streptavidins had biotin-binding activity, the stability and solubility of rsavC tetraunits were much better than those of rsavS and rsavF, indicating that signal peptide and/or extra amino acid residues in rsavS and rsavF have negative effects on streptavidin. Meanwhile, the signal peptide and extra amino acid residues in rsavS and rsavF made it difficult for polypeptides to fold into functional proteins. After refolding of denaturing-purified proteins in vitro, both the specific activities and biotin binding sites of renatured streptavidins were 1.4-times as that of proteins obtained by native Ni-NTA purification. Because the denaturing-purified rsavC is easy of refolding into functional protein, the better strategy for production of active rsavC is to isolate the protein from IPTG-induced E. coli extracts by denaturing Ni-NTA affinity chromatography followed by refolding of purified polypeptide in vitro.
Liu
Xipeng
X
College of Life Sciences & Technology, Shanghai Jiaotong University, 1954 Hua-Shan Road, 200030, Shanghai, China.
Liu
Jianhua
J
D001665:Binding Sites; D001710:Biotin / Q000737:chemistry*; D019921:Biotinylation; D002845:Chromatography; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017510:Protein Folding; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry*; D013997:Time Factors; D014462:Ultrafiltration
D010455:Peptides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D001710:Biotin; D019809:Streptavidin