Bacterial expression, purification, and in vitro N-myristoylation of fusion hepatitis B virus preS1 with the native-type N-terminus.
Very low-level expression of hepatitis B virus (HBV) preS1 with the native-type N-terminus hampered the biochemical and functional studies on its myristoylation. In the present study, the fusion HBV preS1 with the native-type N-terminus and a His6-Tag fused to C-terminus (HBV preS1-HT) was highly expressed in Escherichia coli. This was due to an introduced mutation of the rare codon GGA found in the HBV preS1 to the codon preferred by E. coli, GGU. The protein was rapidly purified from bacterial lysate by Ni-IDA affinity chromatography. The experimental assays using 3H-labeled substrate demonstrate that the purified HBV preS1-HT can be effectively N-myristoylated by recombinant human protein N-myristoyltransferase (NMT) in vitro.
Ma
Han-Hui
HH
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Yang
Li
L
Yang
Xin-Ying
XY
Xu
Zheng-Ping
ZP
Li
Bo-Liang
BL
D000217:Acyltransferases / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006514:Hepatitis B Surface Antigens / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006515:Hepatitis B virus / Q000235:genetics*; D006639:Histidine; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019814:Myristic Acid / Q000378:metabolism*; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C050806:presurface protein 1, hepatitis B surface antigen; D019814:Myristic Acid; D006639:Histidine; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase