Cloning, expression, purification, and characterization of zebrafish cytosolic serine hydroxymethyltransferase.
A cDNA which encodes for zebrafish serine hydroxymethyltransferase (SHMT) has been cloned into a pET43.1a vector as a NdeI-EcoRI insert and transformed into HMS174(DE3) cells. After induction with isopropyl thiogalactoside, the enzyme was purified with a three-step purification protocol and about 15 mg of pure enzyme was obtained per liter of culture. Spectral and structural characteristics of the recombinant zebrafish SHMT are similar to the rabbit and human cytosolic SHMT. Kinetic constants for the natural substrates l-serine and tetrahydrofolate are also comparable to the values obtained previously for the rabbit and human cytosolic enzyme.
Chang
Wen-Ni
WN
Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, School of Medicine, Tainan 701, Taiwan.
Tsai
Jen-Ning
JN
Chen
Bing-Hung
BH
Fu
Tzu-Fun
TF
D000818:Animals; D003593:Cytoplasm / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D015870:Gene Expression; D012696:Glycine Hydroxymethyltransferase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D017386:Sequence Homology, Amino Acid; D015027:Zebrafish / Q000235:genetics; D029961:Zebrafish Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D011994:Recombinant Proteins; D029961:Zebrafish Proteins; D012696:Glycine Hydroxymethyltransferase