Selecting an appropriate method for expressing S locus F-box-S2 recombinant protein

Area of Science:

• Plant reproductive biology
• Molecular genetics
• Biochemistry

Background:

• Gametophytic self-incompatibility (GSI) in apples is controlled by the S locus, involving female and male determinants.
• The structure of the female determinant (S-RNase) is known, but the male determinant (S-locus F-box, SLF/SFB) structure remains elusive due to expression challenges.

Purpose of the Study:

• To evaluate various in vivo and in vitro expression systems for efficient recombinant expression of apple SLF2.
• To establish a reliable method for producing SLF recombinant proteins for further studies.

Main Methods:

• Screening of prokaryotic and eukaryotic expression systems for apple SLF2 expression.
• Optimization of gene synthesis and culture conditions (high salt, heat shock) in E. coli.
• Quantification of expressed SLF2 protein.

Main Results:

• Successful expression of apple SLF2 was achieved in E. coli.
• The optimized method yielded 1 mg/ml of SLF2 protein.
• Specific conditions involving synthesized genes, high salt culture, and heat shock prior to induction were key.

Conclusions:

• An efficient approach for expressing S-locus F-box (SLF) recombinant proteins has been developed.
• This method overcomes previous expression difficulties and enables future functional and structural analyses of the apple GSI male determinant.