Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Charge engineering of a protein domain to allow efficient ion-exchange recovery
KTH, Superseded Departments, Biochemistry and Biotechnology.ORCID iD: 0000-0002-5391-600X
KTH, Superseded Departments, Biochemistry and Biotechnology.ORCID iD: 0000-0001-8993-048X
KTH, Superseded Departments, Biochemistry and Biotechnology.ORCID iD: 0000-0003-4214-6991
Show others and affiliations
2000 (English)In: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, Vol. 13, no 10, 703-709 p.Article in journal (Refereed) Published
Abstract [en]

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 (Z(wt)) was used asa scaffold when constructing two mutants, Z(basic1) and Z(basic2), with high positive surface charge. Far-ultraviolet circular dichroism measurements showed that they have a secondary structure content comparable to the parental molecule Z(wt). Although melting temperatures (T-m) 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 Z(basic1) and Z(basic2) 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 Z(basic2) and the acidic human serum albumin binding domain (ABD), derived from streptococcal protein G, was also constructed. The gene product Z(basic2)-ABD could be purified using cation-exchange chromatography from a whole cell lysate to more than 90% purity.

Place, publisher, year, edition, pages
2000. Vol. 13, no 10, 703-709 p.
Keyword [en]
circular dichroism, ion-exchange chromatography, molecular modelling, pI, protein A, bacterial receptor domain, escherichia-coli k-12, fusion protein, binding domain, nucleic-acids, force-field, purification, dna, resolution, sequence
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-13275DOI: 10.1093/protein/13.10.703ISI: 000165995500006OAI: oai:DiVA.org:kth-13275DiVA: diva2:323036
Note
QC 20100609Available from: 2010-06-09 Created: 2010-06-09 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Protein Engineering by Directed Evolution and Rational Design
Open this publication in new window or tab >>Protein Engineering by Directed Evolution and Rational Design
2001 (English)Doctoral thesis, comprehensive summary (Other scientific)
Place, publisher, year, edition, pages
Stockholm: KTH, 2001. 87 p.
Keyword
ion-exchange chromatography, subtilisin, Klenow DNA polymerase, Taq DNA polymerase, directed evolution, rational design, charge engineering, phosphonylating inhibitor, phage display, circular dichroism, protein A
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-3171 (URN)91-7283-102-2 (ISBN)
Public defence
2001-06-01, 00:00
Note
QC 20100609Available from: 2001-05-31 Created: 2001-05-31 Last updated: 2010-06-09Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full text

Authority records BETA

Gräslund, TorbjörnUhlén, MathiasNygren, Per-ÅkeHober, Sophia

Search in DiVA

By author/editor
Gräslund, TorbjörnUhlén, MathiasNygren, Per-ÅkeHober, Sophia
By organisation
Biochemistry and Biotechnology
In the same journal
Protein Engineering
Biological Sciences

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 49 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf