Cloning,Prokaryotic Expression And Enzymatic Properties Of Ldha Genes From Four Rodents

Objective:Lactate dehydrogenase(LDH)is an enzyme that catalyzes the reversible conversion of lactate to pyruvate in vivo.It is mainly involved in anaerobic oxidation and gluconeogenesis in normal cells,as well as in aerobic glycolysis in cancer cells.Therefore,it is considered to be an potential target in the treatment of cancer.LDHs are expressed in all organisms except for viruses.LDHs are a tetrameric protein formed by the assembly of four subunits,which are encoded by LDHA,LDHB,LDHC and LDHD genes in mammals.Among them,LDHC is mainly expressed in testicular tissue,and LDH encoded by LDHD only catalyzes the metabolism of D-type lactate.LDH1(H₄),LDH2(H₃M₁),LDH3(H₂M₂),LDH4(H₁M₃)and LDH5(M₄)isozymes are mainly composed of M and H protein subunits encoded by LDHA and LDHB genes respectively.LDHs play an important role in the cell and tissue metabolism,however,there are few studies on Ldha in rodents.At present,there are Ldha and Ldhb reference sequences of Rattus norregicus and Mus musculus in Gen Bank database,however,Cavia procellus only have the predicted sequence of this gene.As far as now,there is no sequence of nucleotide and amino acid of LDH in Rattus tanezumi.In this study,we aimed to clone the encoding sequence of Ldha and construct the recombinant prokaryotic expression plasmids.After the recombinant plasmids were transformed into E.coli,and then LDH proteins were purified.Besides,we also analyze its enzyme activity.It will lay a foundation for further study of LDH including structural characteristics,key amino acids,and screening of inhibitors.Methods:In this study,total RNA was extracted from the thigh muscle of four species of rodents,and the coding sequence of Ldha were amplified by using PCR.The Ldha was cloned into p GM-T vector,and then subcloned into the prokaryotic expression vector p ET,after the results of restriction enzyme digestion and sequencing confirmed that the clone was successful.At the same time,bioinformatics analyse the coding sequences of Ldha among four species of rodents.Then,the constructed prokaryotic expression vector was introduced into E.coil BL21(DE3)and induced by IPTG.The protein expression was purified by nickel affinity column,and the protein was clearly detectable by western blotting.Finally,the enzyme activity of the purified protein was detected by use agarose gel electrophoresis of LDHs method.The enzyme specific activity,optimum pH,pH stability,temperature stability,Km and Vmax of the purified protein to different substrates were detected by continuous monitoring method.Results:1.Cloning and construction of prokaryotic expression vectors ofLdha genes from four species of rodentsThe coding sequences of the four species of rodents Ldha genes were cloned and sequenced,and the lengths of the four species of rodents Ldha genes were all 999 bp.Four recombinant prokaryotic expression plasmids of rodents Ldha were constructed by subcloning the cloned sequence into p ET series prokaryotic expression vectors.2.Bioinformatics analysis of Ldha genes in four species of rodentsBioinformatics analysis showed that the four kind of cloned Ldha genes all encoded 332 amino acids.The theoretical molecular weight is about 36.5 k D.The number of basic amino acids is slightly more than that of acidic amino acids.LDHA protein is hydrophobic and heat-stable protein.The four kind of Ldha are highly similar,and the amino acids at the main action sites are highly conserved.Only 23 amino acid sites may have different degrees of difference.In particular,the Rattus tanezumi has a non-polar glycine at position 229 and the Rattus norregicus is acidic aspartic acid,while Mus musculus and Cavia procellus are acidic glutamic acid.This amino acid site belongs to the substrate binding region.This amino acid difference may lead to differences in its enzymatic properties.3.Optimization of induced expression of Ldha genes in four species of rodentsThe recombinant expression plasmids of four species rodent Ldha genes can be induced by IPTG.In order to obtain more soluble protein,the optimum temperature was 30?at the concentration of 100?mol/L for 6 h.Under this condition,the four expressed proteins were not only large in quantity,but also soluble proteins accounted for more than 30%of the total expressed proteins.4.Purification and identification of LDHA proteins from four species of rodentsFour species of rodents Ldha genes expression proteins were purified by nickel affinity column,and identified as purified proteins by SDS-PAGE electrophoresis,and their molecular weights were consistent with the target protein.Then western blot identified these purified proteins as LDHA proteins.5.Determination of LDHA protein specific activity and enzyme spectrum analysis from four species of rodentsUnder the conditions of pH 7.3 and 25?,the enzymatic specific activities of LDHA purified from the Rattus tanezumi,Rattus norregicus,Mus musculus and Cavia procellus(Conversion of pyruvate to lactate)were 113.76±1.463 U/mg,575.89±5.426 U/mg,311.25±33.420 U/mg and365.32±48.892 U/mg,and the enzymatic activities of the enzymes(Conversion of lactate to pyruvate)were 2.18±0.125 U/mg,4.78±1.375U/mg,1.93±0.788 U/mg and 1.88±0.379 U/mg,repectively.The above results showed that the LDHA protein expression of the four species of rodents encoded by Ldha was more likely to catalyze pyruvate to lactate.In addition,agarose gel electrophoresis of lactate dehydrogenase isozyme analysis showed that the four purified proteins all had catalytic activity and only one isoenzyme band.Because the p I values of the four purified proteins were different,the electrophoresis of these isoenzymes was different.6.Enzymatic properties of LDHA proteins from four species of rodentsIn order to detect the enzyme activity under the condition of different pH value of purified protein,we selected a pH value for every 0.5increase in the range of pH 5.0-10.0,and used three kinds of buffer solutions to prepare a total of 11 buffer solutions with different pH values.The results showed that the optimal pH of LDHA purified protein from Rattus tanezumi,Rattus norregicus and Mus musculus were 7.0,while the Cavia procellus was 6.0.If the four LDHA purified proteins were mixed with the above 11 pH buffer solutions in a ratio of 1:4 and placed them at25?for 1 hour.Then we detected the enzyme activity under the optimal pH conditions.The results showed that the four purified proteins mixed with pH 7.0 buffer solution had the least effects on enzyme activity.In addition,more than 80%of the enzyme activity of the four LDHA purified proteins could be retained with Rattus tanezumi at pH values of5.0-9.0,Rattus norregicus and Mus musculus at pH values of 5.0-10.0,and Cavia procellus at pH values of 5.5-7.5.The four purified proteins were stored at different temperatures of 4?,25?,30?,35?,40?,45?and 50?for 1 h,respectively.Rattus tanezumi,Rattus norregicus and Mus musculus retained more than 90%of the enzymatic activity at4?-40?,while Cavia procellus retained more than 90%of the enzymatic activity at 4?-30?.Under the optimum pH conditions and25?,the LDHA protein Km were 0.17±0.193 mmol/L,0.247±0.033mmol/L,0.195±0.158 mmol/L and 0.224±0.028 mmol/L towards pyruvate in Rattus tanezumi,Rattus norregicus,Mus musculus and Cavia procellus,respectively.The Km were 0.088±0.008 mmol/L,0.136±0.028mmol/L,0.058±0.0006 mmol/L and 0.094±0.016 mmol/L towards NADH,respectively.The Km were 22.54±0.007 mmol/L,27.042±4.095mmol/L,21.21±1.961 mmol/L and 51.57±15.58 mmol/L towards lactate,respectively.The Km were 0.413±0.069mmol/L,0.193±0.028mmol/L,0.39±0.064mmol/L and 0.711±0.298mmol/L towards NAD⁺respectively.Conclusions:1.The coding sequences of the Ldha genes were cloned and expressed from Rattus tanezumi,Rattus norregicus,Mus musculus and Cavia procellus.Then we detected the enzyme activity from four kinds of Ldha prokaryotic expression purified protein.The result showed that the activity of catalyzing the conversion of pyruvate to lactic acid is much higher than that of catalyzing the conversion of lactic acid to pyruvate.2.It is the first time to clone the Ldha of Rattus tanezumi and submit the sequence to NCBI Gen Bank database.The registration number is MW201965.Bioinformatics analysis showed that the coding sequence of Ldha in Rattus tanezumi was very similar to Rattus norregicus,and the biological evolution analysis also showed that the genetic relationship between them had a close relationship.However,the difference of amino acid 229 between them might be reason why there was a difference in their enzymatic properties.The Cavia procellus had the farthest genetic relationshipand the greatest difference in amino acid sequences compared with the other three kinds of rodents,which may lead to the great differences in the enzymatic properties such as the optimal pH value,pH stability and temperature stability.