Preliminary Crystallographic Study Of Transcription Factors XylR2,CodY And TcsR2 In Lactococcus Lactis

Lactic acid bacteria are widely used in food,medicine,etc.However,the adverse conditions encountered in the production process often affect the growth of strains and the accumulation of product.The transcription factors in the cells can respond to the changes of external environment by regulating the expression of the target gene,thereby making the strains can quickly resist and adapt to the adverse conditions.Xyl R2,a ROK family transcription factor,is involved in acid resistance and xylose utilization.In our previous study,overexpression of Xyl R2 could improve the xylose utilization,acid tolerance,and Nisin yield of the strains.The global transcription factor Cod Y is involved in casein degradation,peptide and amino acid transportation,carbon and nitrogen metabolism,and virulence genes regulation.It regulates the expression of more than 100 genes.Lux R family transcription factor Tcs R2 has never been reported,and its homologs Ces R and Lia R are involved in acid stress response and the antibiotics tolerance of the strains.Our previous study found that the above three transcription factors may be involved in the acid stress response of strains.However,their crystal structures in Lactococcus lactis have not yet been resolved,especially Xyl R2,even the crystal structure of its homologous proteins have not been reported.In this article,preliminary crystallography of transcription factors Xyl R2,Cod Y and Tcs R2 in Lactococcus lactis F44 was studied.Heterologous expression strain of Xyl R2 was constructed by molecular biology methods,and high-purity Xyl R2 protein was obtained through expression and purification.The initial crystallization conditions suitable for Xyl R2 were explored by crystallographic methods,and the high-quality Xyl R2 crystal was obtained by optimization.X-ray synchrotron radiation was used to diffract Xyl R2 crystal,and the diffraction data were collected and preliminary processed.Since the phase cannot be determined by molecular replacement during the analysis,Xyl R2 was subsequently labeled with selenomethionine.The crystallization conditions of selenium-labeled Xyl R2 and the selenium-labeled Xyl R2-nucleic acid complex were explored and optimized to obtain high-quality selenoprotein crystals and co-crystals,respectively.The corresponding data were collected by X-ray synchrotron radiation.The crystallographic preliminary study of Cod Y and Tcs R2 was carried out by the same method,and the optimized conditions and crystal diffraction data of Cod Y were obtained.The high-purity Tcs R2 protein was obtained,while the full-length Tcs R2 could not be crystallized due to the complexity of its own structure.The further research of Tcs R2 could be done through protein truncation.A XylR2 crystal with a resolution of 2.6 A was obtained under the condition of protein concentration of 4.8 mg/m L,0.2 M ammonium citrate dibasic,20%polyethylene glycol 3350,p H 5.1.Preliminary data processing using HKL3000 and CCP4 showed that Xyl R2 belongs to the P222 space group and exists as a dimer.Selenium-labeled Xyl R2 crystal with a resolution of 2.8 ? was obtained at a protein concentration of 3.8 mg/m L,0.2 M ammonium citrate dibasic,22% polyethylene glycol 3350,p H 5.0.Complex co-crystallization with a resolution of 2.6 ? was obtained at 3.5 mg/m L,protein and nucleic acid molar ratio of 1: 1.2,0.2 M ammonium citrate dibasic,20% polyethylene glycol 3350,p H 5.2,the additive 3.0%ethanol.A Cod Y crystal with a resolution of 4.5 ? was obtained at a protein concentration of 2.0 mg/m L,0.2 M Sodium phosphate monobasic monohydrate,20%polyethylene glycol 3350,p H 4.7.Through preliminary crystallographic studies of three transcription factors,crystallization-related data were obtained,which laid the foundation for subsequent analysis of protein structure and exploration of the regulatory mechanism in cells.