An encoded N-terminal extension results in low levels of heterologous protein production in Escherichia coli

Table 2 X. nematophila tdk plasmid design and resulting Tdk protein levels

Oligonucleotide name	Oligonucleotide sequence (5'->3') ^a	Resulting plasmid name	N-terminus of engineered protein^b	Relative amount of protein expressed (% of short-form) ^c
NcoI Tdkshort	CCATGG CTCAGCTTTATTTTTAT	pETSTdk	M AQLY...	100
NcoI Tdklong	CCATGG ACGGGCCAATGGCTCAG	pETLTdk	MDGPMAQLY...	0
NcoI MDGATdk	CCATGG ACGGGGCAATGGCTCAG	pETMDGATdk	MDGAMAQLY...	0
NcoI MDPGTdk	CCATGG ACCCAGGGATGGCTCAGCTTTATTTTTATTATTCTGC	pETMDPGTdk	MDPGMAQLY...	0
NcoI MDG(GGU)PTdk	CCATGG ACGGTCCAATGGCTCAG	pETMDG(GGU)PTdk	MDGPMAQLY...	0
NcoI MAAGTdk	CCATGG CCGCCGGGATGGCTCAGCTTTATTTTTATTATTCTG	pETMAAGTdk	M AAGMAQLY...	0
NcoI MDAGTdk	CCATGG ACGCCGGGATGGCTCAGCTTTATTTTTATTATTCTG	pETMDAGTdk	MDAGMAQLY...	0.2
NcoI MAGPTdk	CCATGG CCGGGCCAATGGCTCAG	pETMAGPTdk	M AGPMAQLY...	1
NcoI MGPDTdk	CCATGG GGCCAGACATGGCTCAGCTTTATTTTTATTATTCTGC	pETMGPDTdk	M GPDMAQLY...	13
NcoI MAAAGTdk	CCATGG CCGCCGCCGGGATGGCTCAGCTTTATTTTTATTATTCTG	pETMAAAGTdk	M AAAGMAQLY...	15
NcoI MAPGDTdk	CCATGG CCCCAGGGGACATGGCTCAGCTTTATTTTTATTATTCTG	pETMAPGDTdk	M APGDMAQLY...	47
NcoI MGDPTdk	CCATGG GGGACCCAATGGCTCAGCTTTATTTTTATTATTCTGC	pETMGDPTdk	M GDPMAQLY...	77
NcoI MGAATdk	CCATGG GGGCCGCCATGGCTCAGCTTTATTTTTATTATTCTG	pETMGAATdk	M GAAMAQLY...	80
NcoI MAGATdk	CCATGG CCGGGGCCATGGCTCAGCTTTATTTTTATTATTCTG	pETMAGATdk	M AGAMAQLY...	88
NcoI MDAPTdk	CCATGG ACGCGCCAATGGCTCAG	pETMDAPTdk	MDAPMAQLY...	93
NcoI MAPDGTdk	CCATGG CCCCAGACGGGATGGCTCAGCTTTATTTTTATTATTCTG	pETMAPDGTdk	M APDGMAQLY...	103

^aUnderlined nucleotides indicate engineered restriction sites used in cloning ^bAn italicized M indicates an N-terminal f-Met residue that, based on the identity of the second amino acid residue, is predicted to be posttranslationally removed by PepM ^cAmount of Tdk protein detected by immunoblot analysis of E. coli KY895 carrying pTara and the pET-28a(+)-derived plasmid listed and expressed relative to the level of detected short-form Tdk and background corrected against the pET-28a(+) vector-only level.

ISSN: 1475-2859