Details
| Original language | English |
|---|---|
| Pages (from-to) | 73-77 |
| Number of pages | 5 |
| Journal | Applied Microbiology and Biotechnology |
| Volume | 42 |
| Issue number | 1 |
| Publication status | Published - Oct 1994 |
| Externally published | Yes |
Abstract
The influence of different N-terminal affinity fusion domains on the product heterogeneity of recombinant proteins expressed in Escherichia coli was investigated. N-Terminal extended forms of the restriction endonuclease EcoRV with either glutathione-S-transferase [GST], histidine hexapeptide [(His)6], or a combination of GST and (His)6 [GST-(His)6] were compared to native EcoRV with respect to expression level, susceptability to inclusion body formation and protein fragmentation. Fingerprinting of product heterogeneity was done by using two-dimensional (2-D) non equilibrium pH-gradient electrophoresis with subsequent immunoblotting. Fusion proteins containing GST were poorly expressed compared to native EcoRV. In addition, GST fusion proteins were highly susceptible to invivo aggregation and fragmentation and displayed more heterogeneity on 2-D immunoblots. However, the sole presence of oligohistidine at the N-terminus of EcoRV proved to be advantageous. Fragmentation of (His)6-EcoRV was not observed and 2-D immunoblots did not show heterogenous forms of the recombinant protein. In addition, fusion of the histidine-hexapeptide to the N-terminus of native EcoRV increased the expression level of the recombinant protein twofold compared to native EcoRV. Inclusion body formation of the (His)6-EcoRV fusion protein was intensive when cells were grown at 37°C but not at 30°C. The advantage of oligohistidine fusion to EcoRV was finally demonstrated by purifying soluble (His)6-EcoRV in a single-step procedure from crude cell lysates using immobilized metal chelate affinity chromatography.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
- Immunology and Microbiology(all)
- Applied Microbiology and Biotechnology
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In: Applied Microbiology and Biotechnology, Vol. 42, No. 1, 10.1994, p. 73-77.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Comparison of N-terminal affinity fusion domains
T2 - effect on expression level and product heterogeneity of recombinant restriction endonuclease EcoRV
AU - Oswald, T.
AU - Wende, W.
AU - Pingoud, A.
AU - Rinas, U.
PY - 1994/10
Y1 - 1994/10
N2 - The influence of different N-terminal affinity fusion domains on the product heterogeneity of recombinant proteins expressed in Escherichia coli was investigated. N-Terminal extended forms of the restriction endonuclease EcoRV with either glutathione-S-transferase [GST], histidine hexapeptide [(His)6], or a combination of GST and (His)6 [GST-(His)6] were compared to native EcoRV with respect to expression level, susceptability to inclusion body formation and protein fragmentation. Fingerprinting of product heterogeneity was done by using two-dimensional (2-D) non equilibrium pH-gradient electrophoresis with subsequent immunoblotting. Fusion proteins containing GST were poorly expressed compared to native EcoRV. In addition, GST fusion proteins were highly susceptible to invivo aggregation and fragmentation and displayed more heterogeneity on 2-D immunoblots. However, the sole presence of oligohistidine at the N-terminus of EcoRV proved to be advantageous. Fragmentation of (His)6-EcoRV was not observed and 2-D immunoblots did not show heterogenous forms of the recombinant protein. In addition, fusion of the histidine-hexapeptide to the N-terminus of native EcoRV increased the expression level of the recombinant protein twofold compared to native EcoRV. Inclusion body formation of the (His)6-EcoRV fusion protein was intensive when cells were grown at 37°C but not at 30°C. The advantage of oligohistidine fusion to EcoRV was finally demonstrated by purifying soluble (His)6-EcoRV in a single-step procedure from crude cell lysates using immobilized metal chelate affinity chromatography.
AB - The influence of different N-terminal affinity fusion domains on the product heterogeneity of recombinant proteins expressed in Escherichia coli was investigated. N-Terminal extended forms of the restriction endonuclease EcoRV with either glutathione-S-transferase [GST], histidine hexapeptide [(His)6], or a combination of GST and (His)6 [GST-(His)6] were compared to native EcoRV with respect to expression level, susceptability to inclusion body formation and protein fragmentation. Fingerprinting of product heterogeneity was done by using two-dimensional (2-D) non equilibrium pH-gradient electrophoresis with subsequent immunoblotting. Fusion proteins containing GST were poorly expressed compared to native EcoRV. In addition, GST fusion proteins were highly susceptible to invivo aggregation and fragmentation and displayed more heterogeneity on 2-D immunoblots. However, the sole presence of oligohistidine at the N-terminus of EcoRV proved to be advantageous. Fragmentation of (His)6-EcoRV was not observed and 2-D immunoblots did not show heterogenous forms of the recombinant protein. In addition, fusion of the histidine-hexapeptide to the N-terminus of native EcoRV increased the expression level of the recombinant protein twofold compared to native EcoRV. Inclusion body formation of the (His)6-EcoRV fusion protein was intensive when cells were grown at 37°C but not at 30°C. The advantage of oligohistidine fusion to EcoRV was finally demonstrated by purifying soluble (His)6-EcoRV in a single-step procedure from crude cell lysates using immobilized metal chelate affinity chromatography.
UR - http://www.scopus.com/inward/record.url?scp=0028073781&partnerID=8YFLogxK
U2 - 10.1007/BF00170227
DO - 10.1007/BF00170227
M3 - Article
C2 - 7765822
AN - SCOPUS:0028073781
VL - 42
SP - 73
EP - 77
JO - Applied Microbiology and Biotechnology
JF - Applied Microbiology and Biotechnology
SN - 0175-7598
IS - 1
ER -