Charge engineering of a protein domain to allow efficient ion-exchange recovery.
We have created protein domains with extreme surface charge. These mutated domains allow for ion-exchange chromatography under conditions favourable for selective and efficient capture, using Escherichia coli as a host organism. The staphylococcal protein A-derived domain Z (Zwt) was used as a scaffold when constructing two mutants, Zbasic1 and Zbasic2, with high positive surface charge. Far-ultraviolet circular dichroism measurements showed that they have a secondary structure content comparable to the parental molecule Zwt. Although melting temperatures (Tm) of the engineered domains were lower than that of the wild-type Z domain, both mutants could be produced successfully as intracellular full-length products in E. coli and purified to homogeneity by ion-exchange chromatography. Further studies performed on Zbasic1 and Zbasic2 showed that they were able to bind to a cation exchanger even at pH values in the 9 to 11 range. A gene fusion between Zbasic2 and the acidic human serum albumin binding domain (ABD), derived from streptococcal protein G, was also constructed. The gene product Zbasic2-ABD could be purified using cation-exchange chromatography from a whole cell lysate to more than 90% purity.
Gräslund
T
T
Department of Biotechnology, Royal Institute of Technology (KTH), S-100 44 Stockholm, Sweden.
Lundin
G
G
Uhlén
M
M
Nygren
P A
PA
Hober
S
S
D001426:Bacterial Proteins / Q000737:chemistry; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004926:Escherichia coli / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D012709:Serum Albumin / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000737:chemistry; D055672:Static Electricity; D013816:Thermodynamics
D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D013205:Staphylococcal Protein A