Expression of Sperm Acrosomal SLLP1 Binding (SAS1B) Protein in a Mammalian System for Crystallization

Abstract

SAS1B (or Ovastacin) is considered an ideal candidate for both female contraception and for cancer therapies. SAS1B first appears in oocytes of follicles at the primary‐secondary transition in pre‐pubertal rodent ovaries and beyond. Quiescent oocytes within primordial and primary follicles are SAS1B negative. In 7 eutherian adult ovaries, the precise temporal and spatial onset of SAS1B expression was conserved. Crystal studies are a vital tool for drug design and for better understanding the molecular structure of targetable proteins, such as SAS1B. Full length SAS1B and its mature form (lacking the N terminus) were PCR‐cloned using gene specific primers sequence containing a kozak sequence at the N‐terminus and six histidine tags at the C‐terminus, using the TOPO cloning strategy. The PCR products were introduced into a pcDNA 3.4 vector and transformed into chemically competent E. Coli cells. Cells were plated on ampicillin infused Luria agar plates and colonies were allowed to grow overnight. Colonies were inoculated in fresh ampicillin containing Luria broth and DNA was extracted using a miniprep kit. Eluted DNA was sent for DNA sequencing. One of the full length clones, clone #9, showed the correct insert for full length SAS1B while screening of E. Coli colonies for the correct sequence for matured SAS1B is ongoing. Subsequently, once the correct sequences have been confirmed through sequencing, transfection of the correct vector‐insert species will be conducted using Expi293 mammalian cells, followed by protein isolation, detection and purification for the purpose of obtaining soluble SAS1B to generate crystals.