Reports of Biochemistry

Background: Inclusion body formation in E. coli is a significant problem in recombinant protein production. The aim of this study was to improve the solubility of recombinant bovine sex determining region Y protein (SRY) in BL21 (DE3) E. coli cells.
Methods: In this research two recombinant bovine SRY (rbSRY) sequences were analyzed; these were wild-type SRY (wtbSRY) and codon-optimized SRY (cobSRY). Their expression in various culture conditions was examined; these differences included IPTG concentrations, temperatures, and media stabilizers.
Results: IPTG and temperature significantly affected rbSRY solubility (P < 0.001). The optimum IPTG concentration and temperatures for wtbSRY and cobSRY induction were 0.3 mM at 27 and 32 °C, respectively. In addition, arginine and sorbitol concentrations significantly affected rbSRY solubility (P < 0.01). Solubility of rbSRY protein was highest from the cobSRY construct in the presence 0.2 M arginine and 0.3 M sorbitol. The highest inclusion body production occurred with high glucose concentrations.
Conclusions: We found that modifications in temperature and IPTG and stabilizer concentrations affected rbSRY solubility.
 

Background: Sex selection of sperm by separating X- and Y-chromosome bearing spermatozoa is critical for efficiently obtaining the desired sex of animal offspring in the livestock industry. The purpose of this study was to produce a goat polyclonal antibody (pAb) against the bovine Sex Determining Region Y
chromosome (bSRY) to separate female- and male-bearing spermatozoa.

Methods: To produce a goat polyclonal antibody against bSRY, a female goat was subcutaneously immunized with 27 kDa of recombinant bSRY (rbSRY) protein as the antigen. The anti-bSRY pAb was purified by ion-exchange chromatography. The purity of the pAb was determined using the SDS-PAGE
method. The biological activity of the anti-bSRY pAb was examined using PCR to assess the binding affinity of pAb for the bSRY antigen and commercially sexed bull sperm.

Results: The total amount of purified anti-bSRY pAb was approximately 650 mg/goat serum (13 mg/mL). Interestingly, our data showed that the binding affinity of our pAb to the Y bearing was high, while the binding affinity of that to the X-chromosome bearing sperm was similar to the negative control.

Conclusions: In conclusion, our findings show that the goat anti-SRY pAb specifically binds to Ychromosome bearing sperm that suggesting its potential use for sex selection.