Recombinant Lactobacillus fermentum ATP synthase subunit b (atpF)
Product Specs
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Target Background
KEGG: lfe:LAF_0437
Q&A
Basic Research Questions
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What is the structural difference between ATP synthase subunit b (atpF) and subunit c (atpE) in Lactobacillus fermentum?
ATP synthase subunit b (atpF) functions as part of the peripheral stalk connecting the membrane (F0) and soluble (F1) sectors of the ATP synthase complex. It typically forms a homodimer and plays a primarily structural role in the rotary catalytic mechanism . The b subunit has a distinctive structure with a transmembrane domain and an extended cytoplasmic region that contains autonomous domains, including a bend sequence (residues 23–26) and two long α-helical regions (residues 34–78, 85–156) separated by a turn sequence (79–84) .
In contrast, ATP synthase subunit c (atpE) is part of the F0 sector and functions as a lipid-binding protein . While the b subunit forms the peripheral stalk, the c subunit forms a ring in the membrane-embedded F0 sector that is essential for proton translocation during ATP synthesis .
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What expression systems are recommended for producing recombinant Lactobacillus fermentum ATP synthase subunit b?
The optimal expression systems for producing recombinant ATP synthase subunits include:
Expression System Advantages Considerations E. coli High yield, economical, rapid expression May lack proper folding for complex proteins Yeast Post-translational modifications, proper folding Longer production time, lower yields Baculovirus Complex protein folding, post-translational modifications More expensive, technically challenging Mammalian cells Most authentic post-translational processing Highest cost, longest production time For Lactobacillus proteins specifically, E. coli is frequently used when proper folding can be achieved, while more complex systems may be necessary if the native structure is difficult to maintain . Selection should be based on the specific experimental requirements and downstream applications.
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How can researchers verify the purity and identity of recombinant ATP synthase subunit b?
A multi-method approach is recommended for comprehensive quality assessment:
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SDS-PAGE analysis to confirm molecular weight and assess initial purity (>90% purity is typically desired)
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Western blotting with specific antibodies against atpF or included tags
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Circular dichroism (CD) spectroscopy to verify the expected secondary structure content (predominantly α-helical for the b subunit)
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Analytical ultracentrifugation to confirm the homodimeric state and assess homogeneity
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Mass spectrometry for precise molecular weight determination and sequence verification
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Limited proteolysis combined with mass spectrometry to verify domain organization
These complementary approaches provide a comprehensive assessment of both purity and structural integrity.
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