Subcloning of prochymosin cDNA for Plac controlled expression

Area of Science:

• Molecular Biology
• Protein Expression
• Enzymology

Background:

• Prochymosin cDNA clones are essential for understanding chymosin's role in dairy and food industries.
• Efficient expression systems are needed to produce active chymosin for various applications.

Purpose of the Study:

• To construct expression plasmids encoding an activable prochymosin zymogen.
• To verify the integrity of reverse transcripts used for expression.
• To characterize the properties of the expressed and activated prochymosin.

Main Methods:

• Utilized overlapping prochymosin cDNA segments to build expression plasmids.
• Employed the pUC9 vector with the lac promoter for controlled gene expression.
• Characterized the active product using milk-clotting assays, caseinography, and immunoprecipitation followed by protein electrophoresis.

Main Results:

• Successfully constructed plasmids expressing an activable prochymosin zymogen.
• The expressed product, after activation, exhibited properties consistent with calf chymosin.
• Confirmed the integrity of the reverse transcripts through functional expression.

Conclusions:

• The developed expression system effectively produces active calf chymosin.
• The constructed plasmids serve as a versatile source of prochymosin cDNA for future expression studies.
• This work validates a method for verifying cDNA integrity via functional protein expression.