Difference between revisions of "Part:BBa K2929000:Experience"
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==iGEM 2019 St Andrews - Part Development== | ==iGEM 2019 St Andrews - Part Development== | ||
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<p>The Human IgG4 CH3 Domain expresses in great quantities from a pEHISTEV vector (~6 mg/mL in 20 mL for 1 L E. coli culture) if expressed in SoluBL21 cells and induced with 0.2 mM IPTG at 25°C overnight.</p> | <p>The Human IgG4 CH3 Domain expresses in great quantities from a pEHISTEV vector (~6 mg/mL in 20 mL for 1 L E. coli culture) if expressed in SoluBL21 cells and induced with 0.2 mM IPTG at 25°C overnight.</p> | ||
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<p style="text-align:center;"><font size=-2>Figure 1. Purification of the human IgG wild type using nickel affinity chromatography and TEV cleavage. The expressed protein is highly soluble and was purified well via this method. The reverse nickel purified fraction only contains one band at the expected 12 kDa mass, so pure recombinant protein was obtained.</p> | <p style="text-align:center;"><font size=-2>Figure 1. Purification of the human IgG wild type using nickel affinity chromatography and TEV cleavage. The expressed protein is highly soluble and was purified well via this method. The reverse nickel purified fraction only contains one band at the expected 12 kDa mass, so pure recombinant protein was obtained.</p> | ||
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<p style="text-align:center;"><font size=-2>Figure 2. Mass-spectrum for the expressed and purified wild type human IgG fragment. The spectrum shows a very strong and clear signal at the expected molecular mass (12373 Da) which correspond to the recombinant protein.</p> | <p style="text-align:center;"><font size=-2>Figure 2. Mass-spectrum for the expressed and purified wild type human IgG fragment. The spectrum shows a very strong and clear signal at the expected molecular mass (12373 Da) which correspond to the recombinant protein.</p> | ||
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===User Reviews=== | ===User Reviews=== | ||
Latest revision as of 14:55, 19 October 2019
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
Applications of BBa_K2929000
iGEM 2019 St Andrews - Part Development
The Human IgG4 CH3 Domain expresses in great quantities from a pEHISTEV vector (~6 mg/mL in 20 mL for 1 L E. coli culture) if expressed in SoluBL21 cells and induced with 0.2 mM IPTG at 25°C overnight.
BBa_K2929000 is mostly soluble and only a small percentage of the total amount of expressed protein is insoluble. As figure 1. shows, highly pure protein is obtainable via His-tagging and nickel immobilized metal affinity chromatography (IMAC), followed by His-tag cleavage and reverse nickel chromatography. The C425F mutation’s influence on solubility is uncertain, but this wild type is much more soluble than any of its mutants.
The expressed protein’s identity was also confirmed by electro-spray mass spectrometry, showing a correct molar mass for this protein (expected mass of 12373.8 Da) (Figure 2.).
Figure 1. Purification of the human IgG wild type using nickel affinity chromatography and TEV cleavage. The expressed protein is highly soluble and was purified well via this method. The reverse nickel purified fraction only contains one band at the expected 12 kDa mass, so pure recombinant protein was obtained.
Figure 2. Mass-spectrum for the expressed and purified wild type human IgG fragment. The spectrum shows a very strong and clear signal at the expected molecular mass (12373 Da) which correspond to the recombinant protein.
User Reviews
UNIQ18bb0a21fac16d23-partinfo-00000001-QINU UNIQ18bb0a21fac16d23-partinfo-00000002-QINU