Heterologous expression of bovine spermadhesin-1 using escherichia coli as biofactory

Abstract

Seminal plasma proteins (SPP) are fundamental for oocyte fertilization by sperm cells. In bovine, the structure and function of SPP have been widely described in several studies, where the spermadhesin family has been highlighted. Spermadhesin proteins are closely related to sperm motility and viability along with protecting the sperm cells against oxidative stress. Spermadhesin-1, also known as acidic Seminal Fluid Proteins (aSFP), exhibits a redox activity that protects the sperm cells from reactive oxygen species (ROS), an important feature that could be taken in advantage to improve the postthaw seminal quality of sperm cells after cryopreservation processes. Therefore, the aim of this research was to produce heterologous aSFP using Escherichia coli as a cell factory. For this purpose, the DNA sequence of aSFP was inserted into a pDAss plasmid, containing a six-histidine tag (6xhis). The obtained construct was used to transform BL21 (DE3) E. coli competent cells. The expression trials were developed at three different temperatures (37°,25°, and 16°C) and two different concentrations of Isopropyl-β-D-1-thiogalactopiranoside (IPTG) as the expression inductor (0.5 y 1.0 mM). The aSFP-H6 produced was purified by affinity chromatography, and the peptide sequence was verified through mass spectrometry. Results evidenced the best conditions for the recombinant aSFP-H6 production (16°C and 1.0 mM of IPTG). In this sense, the viability and the conditions to produce heterologous bovine aSFP were described and could be further used to enhance cryopreservation bovine sperm mediums.