Expression of cDNA fragment encoding sperm membrane peptide in E. coli

Area of Science:

• Molecular Biology
• Biotechnology
• Protein Expression

Background:

• Developing efficient expression systems for recombinant protein production is crucial in biotechnology.
• Secretory expression offers advantages for protein purification and refolding.
• Existing vectors may have limitations in yield and secretion efficiency.

Purpose of the Study:

• To construct and characterize a novel secretory high-level expression cloning vector, pSBC-20.
• To demonstrate the vector's capability for secreting foreign peptide fragments.
• To evaluate the yield of secreted recombinant protein.

Main Methods:

• Construction of the pSBC-20 vector by inserting the ompA signal peptide into the pBV 220 vector.
• Ligation of a human sperm protein fragment cDNA into the pSBC-20 polylinker, creating pRSD-220.
• Transformation of E. coli with pRSD-220 and induction of protein expression at 42°C.

Main Results:

• The pSBC-20 vector enables secretion of cloned foreign genes into the periplasmic space or medium.
• Recombinant plasmid pRSD-220 successfully directed the secretion of a human sperm protein fragment.
• A high secretion rate was observed, yielding approximately 60 mg of gene product per liter of culture medium.

Conclusions:

• The pSBC-20 vector is an effective tool for high-level secretory expression of foreign proteins in E. coli.
• The system demonstrates significant potential for producing therapeutic or research proteins.
• Optimized conditions led to substantial yields of secreted recombinant peptides.