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Molecular cloning and nucleotide sequence of cDNA coding for calf preprochymosin
Nucleic Acids Research
|April 10, 1982
Summary
Researchers synthesized complementary DNA (cDNA) for calf prochymosin, identifying clones with the complete prochymosin mRNA sequence. This reveals prochymosin
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Calf prochymosin is a precursor to chymosin, a key enzyme in cheesemaking.
- Understanding prochymosin's genetic sequence is crucial for recombinant production and protein engineering.
Purpose of the Study:
- To synthesize and clone complementary DNA (cDNA) encoding calf prochymosin.
- To determine the complete nucleotide sequence of calf prochymosin mRNA.
- To analyze the predicted amino acid sequence and molecular weight of prochymosin and chymosin.
Main Methods:
- Synthesis of DNA complementary to calf stomach mRNA.
- Insertion of cDNA into the pAT153 plasmid using G-C tailing.
- Colony hybridization using a chemically synthesized oligodeoxynucleotide primer.
- Nucleotide sequencing of identified clones.
Main Results:
- Two clones containing the complete calf prochymosin mRNA sequence were identified.
- The nucleotide sequence confirmed the predicted amino acid sequence of calf prochymosin.
- Calculated molecular weights: prochymosin (40431 Da) and chymosin (35612 Da).
Conclusions:
- Calf prochymosin is synthesized as a preprochymosin precursor.
- A 16-amino acid hydrophobic leader sequence was identified, characteristic of secreted proteins.
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