Initiation of translation at an upstream non-AUG codon accounting for N-terminally extended minor forms of recombinant proteins expressed in insect cells.
When the 34 kDa kinase domain of human spleen tyrosine kinase (Syk-KD) was expressed as a C-terminally His-tagged protein in baculovirus-infected Sf-21 insect cells, the purified protein included two forms that migrated slightly differently in SDS-polyacrylamide gel electrophoresis. Intact mass analysis and LC-MS/MS peptide mapping showed that the major and faster-migrating product had the intended amino-acid sequence and 0-6 phosphorylations. This material accounted for about 95% of the purified protein. The minor product was Syk-KD with a 26 amino-acid N-terminal extension. The result suggested the existence of an upstream alternative site for the initiation of translation, and this proved to be an ACG codon derived from the pBacPAK9 vector used to express Syk-KD. The ACG codon was preceded and followed by Kozak-type sequence elements (a purine in the -3 position and a G in the +4 position) that would have enhanced the viability of initiation at ACG. The initiating amino-acid residue was Met for both minor and major products, and both forms of the protein were α-N-acetylated. For the minor product, protein intact mass analysis and peptide mapping both gave results in agreement with the sequence predicted from the DNA. A similar result with the same underlying cause was obtained with insect cell expression of full-length Syk. It appears that similar results are possible whenever this vector is used.
Geoghegan
Kieran F
KF
PharmaTherapeutics Research, Pfizer Inc., Groton, CT 06340, USA. kieran.f.geoghegan@pfizer.com
Feng
Xidong
X
Chang
Jeanne S
JS
Kelleher
Kerry
K
Wu
Paul W
PW
Lin
Laura
L
Rajamohan
Francis
F
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D018387:Codon, Initiator; D005822:Genetic Vectors; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D014176:Protein Biosynthesis; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry; D020539:Sequence Analysis, Protein; D018411:Spodoptera / Q000235:genetics / Q000378:metabolism*; D000072377:Syk Kinase
D018387:Codon, Initiator; D047908:Intracellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase