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  • 1.
    Hammarström, Martin
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Woestenenk, Esmeralda
    KTH, Skolan för bioteknologi (BIO).
    Hellgren, Niklas
    KTH, Skolan för bioteknologi (BIO).
    Härd, Torleif
    Berglund, Helena
    KTH, Skolan för bioteknologi (BIO).
    Effect of N-terminal solubility enhancing fusion proteins on yield of purified target protein2006Ingår i: Journal of Structural and Functional Genomics, ISSN 1345-711X, E-ISSN 1570-0267, Vol. 7, nr 1, s. 1-14Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have studied the effect of solubilising N-terminal fusion proteins on the yield of target protein after removal of the fusion partner and subsequent purification using immobilised metal ion affinity chromatography. We compared the yield of 45 human proteins produced from four different expression vectors: three having an N-terminal solubilising fusion protein (the GB1-domain, thioredoxin, or glutathione S-transferase) followed by a protease cleavage site and a His tag, and one vector having only an N-terminal His tag. We have previously observed a positive effect on solubility for proteins produced as fusion proteins compared to proteins produced with only a His tag in Escherichia coli. We find this effect to be less pronounced when we compare the yields of purified target protein after removal of the solubilising fusion although large target-dependent variations are seen. On average, the GB1+His fusion gives significantly higher final yields of protein than the thioredoxin+His fusion or the His tag, whereas GST+His gives lower yields. We also note a strong correlation between solubility and target protein size, and a correlation between solubility and the presence of peptide fragments that are predicted to be natively disordered.

  • 2.
    Woestenenk, Esmeralda A.
    et al.
    KTH, Tidigare Institutioner, Bioteknologi.
    Hammarström, Martin
    KTH, Tidigare Institutioner, Bioteknologi.
    van den Berg, Susanne
    KTH, Tidigare Institutioner, Bioteknologi.
    Härd, Torleif
    KTH, Tidigare Institutioner, Bioteknologi.
    Berglund, Helena
    KTH, Tidigare Institutioner, Bioteknologi.
    His tag effect on solubility of human proteins produced in Escherichia coli: A comparison between four expression vectors2004Ingår i: Journal of Structural and Functional Genomics, ISSN 1345-711X, E-ISSN 1570-0267, Vol. 5, nr 3, s. 217-229Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have compared four different vectors for expression of proteins with N- or C-terminal hexahistidine (His6) tags in Escherichia coli by testing these on 20 human proteins. We looked at total recombinant protein production levels per gram dry cell weight, solubility of the target proteins, and yield of soluble and total protein when purified by immobilized metal ion affinity purification. It was found that, in general, both N- and C-terminal His6 tags have a noticeable negative effect on protein solubility, but the effect is target protein specific. A solubilizing fusion tag was able to partly counteract this negative effect. Most target proteins could be purified under denaturing conditions and about half of the proteins could be purified under physiological conditions. The highest protein production levels and yield of purified protein were obtained from a construct with a C-terminal His tag. We also observe a large variation in cell growth rate, which we determined to be partly caused by the expression vectors and partly by the targets. This variation was found to be independent of the production level, solubility and tertiary structure content of the target proteins.

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