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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

Functional study of the lacI repressor under induced (left bars) and non-induced (right bars) conditions employing pET28a(+)-CelA2, pET28a(+)M1-CelA2, pET22b(+)-BSLA and pET22b(+)M1-BSLA expression systems. Constructs with an M1 label harbor an epMEGAWHOP optimized vector backbone. Enzyme activity levels were determined with the corresponding screening systems in 96-well microtiter plate formats for CelA2 and BLSA. The reported values are the average of three 96-well microtiter plate measurements in which each hydrolase was expressed 8 times per plate and deviations are calculated from the corresponding mean values.
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Figure 3: Functional study of the lacI repressor under induced (left bars) and non-induced (right bars) conditions employing pET28a(+)-CelA2, pET28a(+)M1-CelA2, pET22b(+)-BSLA and pET22b(+)M1-BSLA expression systems. Constructs with an M1 label harbor an epMEGAWHOP optimized vector backbone. Enzyme activity levels were determined with the corresponding screening systems in 96-well microtiter plate formats for CelA2 and BLSA. The reported values are the average of three 96-well microtiter plate measurements in which each hydrolase was expressed 8 times per plate and deviations are calculated from the corresponding mean values.

Mentions: Expression under inducing and non-inducing conditions was performed to analyze whether the lacI repressor contributes to increased production levels. The constructs pET28a(+)-CelA2 and pET22b(+)-BSLA are under the control of lacI repressor. Figure 3 shows under non-induced conditions a significant difference in activity/lipase production in the pET22b(+)-BSLA expression vector: the mutated backbone yielded 0.34 U/mL compared to 0.04 U/mL of the ‘parent’ pET22b(+). The latter result proves that lacI influences lipase production in pET22b(+)M1-BSLA. Sequencing results confirmed two mutations in lacI (see Table 2). An opposite results was found for the pET28a(+)-CelA2 and the pET28a(+)M1-CelA2 expression systems in which under non-induced conditions only very low cellulase activities could be determined (<0.02 U/mL). These results prove that the lacI is an effective repressor even in the optimized vector backbone. Sequencing results confirmed that there are no mutations in the lacI within the pET28a(+)M1-CelA2 expression system (Table 2). In all four constructs the lacI repressor is functional as confirmed by IPTG induction (Figure 3).


Increasing protein production by directed vector backbone evolution.

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

Functional study of the lacI repressor under induced (left bars) and non-induced (right bars) conditions employing pET28a(+)-CelA2, pET28a(+)M1-CelA2, pET22b(+)-BSLA and pET22b(+)M1-BSLA expression systems. Constructs with an M1 label harbor an epMEGAWHOP optimized vector backbone. Enzyme activity levels were determined with the corresponding screening systems in 96-well microtiter plate formats for CelA2 and BLSA. The reported values are the average of three 96-well microtiter plate measurements in which each hydrolase was expressed 8 times per plate and deviations are calculated from the corresponding mean values.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Functional study of the lacI repressor under induced (left bars) and non-induced (right bars) conditions employing pET28a(+)-CelA2, pET28a(+)M1-CelA2, pET22b(+)-BSLA and pET22b(+)M1-BSLA expression systems. Constructs with an M1 label harbor an epMEGAWHOP optimized vector backbone. Enzyme activity levels were determined with the corresponding screening systems in 96-well microtiter plate formats for CelA2 and BLSA. The reported values are the average of three 96-well microtiter plate measurements in which each hydrolase was expressed 8 times per plate and deviations are calculated from the corresponding mean values.
Mentions: Expression under inducing and non-inducing conditions was performed to analyze whether the lacI repressor contributes to increased production levels. The constructs pET28a(+)-CelA2 and pET22b(+)-BSLA are under the control of lacI repressor. Figure 3 shows under non-induced conditions a significant difference in activity/lipase production in the pET22b(+)-BSLA expression vector: the mutated backbone yielded 0.34 U/mL compared to 0.04 U/mL of the ‘parent’ pET22b(+). The latter result proves that lacI influences lipase production in pET22b(+)M1-BSLA. Sequencing results confirmed two mutations in lacI (see Table 2). An opposite results was found for the pET28a(+)-CelA2 and the pET28a(+)M1-CelA2 expression systems in which under non-induced conditions only very low cellulase activities could be determined (<0.02 U/mL). These results prove that the lacI is an effective repressor even in the optimized vector backbone. Sequencing results confirmed that there are no mutations in the lacI within the pET28a(+)M1-CelA2 expression system (Table 2). In all four constructs the lacI repressor is functional as confirmed by IPTG induction (Figure 3).

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