Cloning And Expression Of Cystathionine ?-lyase And Its Enzymatic Propertirs

Cystathionine?-lyase?CBL?is a key enzyme in the methionine biosynthesis pathway.It can catalyze cystathionine's decomposition into homocysteine?Hcy?,pyruvate and ammonia.The determination of Hcy in plasma can help diagnose the diseases such as the lack of folic acid or vitamin B₁₂ and cardiovascular.At present,one of the popular detection methods for Hcy is Enzymatic cycling assay because it is quick,simple and accurate.CBL plays a key role in it.So it is significant to clone and express CBL with high activity and apply them to this assay.By searching in NCBI,the CBL gene from E.coli K12 was chosed as the target gene.The gene was amplified by PCR,connected with the plasmid vector pET-28a?+?,and transformed into the expression host E.coli BL21 Gold?DE3?plysS.The resulting recombinant was named pET-28a-CBL.By IPTG induction,it successfully expressed a soluble protein,which was about 42 kDa and showed high CBL activity.After the induction by IPTG,the recombinant cell was disrupted by ultrasonic.The supernatant was purified by Ni affinity chromatography using Fusion protein purification kits MagExtractor-His-tag.A electrophoretic pure protein was obtained.Its specific CBL activity was58.24 U/mg.The recovery rate was 48%,and the purification fold was3.66 times.The optimum pH for the recombination CBL was between 8.0 and8.5.The optimum temperature was about 35?.The enzyme was stable at 15³5?,it can keep more than 90%of the initial activity after 4 h incubation at these temperatures.It was the most stable at pH 8.0-8.5.After incubation for 4 h?4??at pH 7.0⁸.5,it can remain more than80%of the initial activity.1 mM of K⁺?Mg²⁺?Fe²⁺?Ca²⁺can improve the activity of the recombinant CBL in different degrees,but Co²⁺,Zn²⁺and Ni²⁺inhibited it.The recombination CBL was applied to Enzymatic cycling assays for Hcy.Using the typical concentrations of three enzymes?CBS=0.014mg/ml,LDH=0.028 mg/ml,CBL=0.04 mg/ml?,the reaction rate had a good linear relationship with the Hcy concentration in the samples.The results demonstrated that the recombination CBL was suitable for Enzymatic cycling assays for Hcy.