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Increasing protein production by directed vector backbone evolution.

Jakob F, Lehmann C, Martinez R, Schwaneberg U - AMB Express (2013)

Bottom Line: The latter demonstrated the general applicability of the epMEGAWHOP method.Protease production using the vector pHY300PLK was increased ~4-times with an average of ~1.25 mutations per kb vector backbone.The epMEGAWHOP does not require any rational understanding of the expression machinery and can generally be applied to enzymes, expression vectors and related hosts. epMEGAWHOP is therefore from our point of view a robust, rapid and straight forward alternative for increasing protein production in general and for biotechnological applications.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, Aachen 52074, Germany. u.schwaneberg@biotec.rwth-aachen.de.

ABSTRACT
Recombinant protein production in prokaryotic and eukaryotic organisms was a key enabling technology for the rapid development of industrial and molecular biotechnology. However, despite all progress the improvement of protein production is an ongoing challenge and of high importance for cost-effective enzyme production. With the epMEGAWHOP mutagenesis protocol for vector backbone optimization we report a novel directed evolution based approach to increase protein production levels by randomly introducing mutations in the vector backbone. In the current study we validate the epMEGAWHOP mutagenesis protocol for three different expression systems. The latter demonstrated the general applicability of the epMEGAWHOP method. Cellulase and lipase production was doubled in one round of directed evolution by random mutagenesis of pET28a(+) and pET22b(+) vector backbones. Protease production using the vector pHY300PLK was increased ~4-times with an average of ~1.25 mutations per kb vector backbone. The epMEGAWHOP does not require any rational understanding of the expression machinery and can generally be applied to enzymes, expression vectors and related hosts. epMEGAWHOP is therefore from our point of view a robust, rapid and straight forward alternative for increasing protein production in general and for biotechnological applications.

No MeSH data available.


Related in: MedlinePlus

The five epMEGAWHOP steps to increase protein production by vector backbone mutagenesis. Step I: megaprimer generation, Step II: amplification of the vector backbone under error-prone conditions (0.05 mM Mn2+), Step III: transformation into E. coli DH5α and isolation of plasmids. The expression hosts (E. coli BL21-Gold (DE3) or B. subtilis DB104) were transformed subsequently with the isolated plasmids, Step IV: agar plate pre-screening with selection based on halo formation, and Step V: screening in microtiter plate format to quantify increase in enzyme production.
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Figure 1: The five epMEGAWHOP steps to increase protein production by vector backbone mutagenesis. Step I: megaprimer generation, Step II: amplification of the vector backbone under error-prone conditions (0.05 mM Mn2+), Step III: transformation into E. coli DH5α and isolation of plasmids. The expression hosts (E. coli BL21-Gold (DE3) or B. subtilis DB104) were transformed subsequently with the isolated plasmids, Step IV: agar plate pre-screening with selection based on halo formation, and Step V: screening in microtiter plate format to quantify increase in enzyme production.

Mentions: Figure 1 shows the scheme of the developed epMEGAWHOP protocol starting from the generation of megaprimers by PCR (Step I). Followed by an amplification of the whole plasmid under error-prone conditions (0.05 mM Mn2+) (Step II) and digestion of the methylated template. In (Step III) the resulting circular DNA is transformed into E. coli DH5α cells. The plasmids are subsequently isolated from E. coli DH5α and transformed into the expression host (E. coli BL21-Gold (DE3) or B. subtilis DB104) and grown on indicator plates (Step IV). Colonies showing halos are transferred into microtiter plates for quantification of enzymatic activity (Step V). Table 1 summarizes the expression constructs used for epMEGAWHOP development.


Increasing protein production by directed vector backbone evolution.

Jakob F, Lehmann C, Martinez R, Schwaneberg U - AMB Express (2013)

The five epMEGAWHOP steps to increase protein production by vector backbone mutagenesis. Step I: megaprimer generation, Step II: amplification of the vector backbone under error-prone conditions (0.05 mM Mn2+), Step III: transformation into E. coli DH5α and isolation of plasmids. The expression hosts (E. coli BL21-Gold (DE3) or B. subtilis DB104) were transformed subsequently with the isolated plasmids, Step IV: agar plate pre-screening with selection based on halo formation, and Step V: screening in microtiter plate format to quantify increase in enzyme production.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750827&req=5

Figure 1: The five epMEGAWHOP steps to increase protein production by vector backbone mutagenesis. Step I: megaprimer generation, Step II: amplification of the vector backbone under error-prone conditions (0.05 mM Mn2+), Step III: transformation into E. coli DH5α and isolation of plasmids. The expression hosts (E. coli BL21-Gold (DE3) or B. subtilis DB104) were transformed subsequently with the isolated plasmids, Step IV: agar plate pre-screening with selection based on halo formation, and Step V: screening in microtiter plate format to quantify increase in enzyme production.
Mentions: Figure 1 shows the scheme of the developed epMEGAWHOP protocol starting from the generation of megaprimers by PCR (Step I). Followed by an amplification of the whole plasmid under error-prone conditions (0.05 mM Mn2+) (Step II) and digestion of the methylated template. In (Step III) the resulting circular DNA is transformed into E. coli DH5α cells. The plasmids are subsequently isolated from E. coli DH5α and transformed into the expression host (E. coli BL21-Gold (DE3) or B. subtilis DB104) and grown on indicator plates (Step IV). Colonies showing halos are transferred into microtiter plates for quantification of enzymatic activity (Step V). Table 1 summarizes the expression constructs used for epMEGAWHOP development.

Bottom Line: The latter demonstrated the general applicability of the epMEGAWHOP method.Protease production using the vector pHY300PLK was increased ~4-times with an average of ~1.25 mutations per kb vector backbone.The epMEGAWHOP does not require any rational understanding of the expression machinery and can generally be applied to enzymes, expression vectors and related hosts. epMEGAWHOP is therefore from our point of view a robust, rapid and straight forward alternative for increasing protein production in general and for biotechnological applications.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, Aachen 52074, Germany. u.schwaneberg@biotec.rwth-aachen.de.

ABSTRACT
Recombinant protein production in prokaryotic and eukaryotic organisms was a key enabling technology for the rapid development of industrial and molecular biotechnology. However, despite all progress the improvement of protein production is an ongoing challenge and of high importance for cost-effective enzyme production. With the epMEGAWHOP mutagenesis protocol for vector backbone optimization we report a novel directed evolution based approach to increase protein production levels by randomly introducing mutations in the vector backbone. In the current study we validate the epMEGAWHOP mutagenesis protocol for three different expression systems. The latter demonstrated the general applicability of the epMEGAWHOP method. Cellulase and lipase production was doubled in one round of directed evolution by random mutagenesis of pET28a(+) and pET22b(+) vector backbones. Protease production using the vector pHY300PLK was increased ~4-times with an average of ~1.25 mutations per kb vector backbone. The epMEGAWHOP does not require any rational understanding of the expression machinery and can generally be applied to enzymes, expression vectors and related hosts. epMEGAWHOP is therefore from our point of view a robust, rapid and straight forward alternative for increasing protein production in general and for biotechnological applications.

No MeSH data available.


Related in: MedlinePlus