Table 1.

Cloning strategy for production of functional XKS1 in E. coli.

Primer (restriction sites in bold; mismatched bases in italic; tags underlined)	Target plasmid	Restriction sites	Resulting plasmid	Resulting strain/XK activity
Fw: 5′-CGCGGATCCTTGTGTT
CAGTAATTCAGAGACAGACAAG-3′
Rev: 5′-CGGGAATTCTTAGATGA
GAGTCTTTTCCAGTTCGCTTAAG-3′
Annealing temperature: 59 °C (30 cycles)	pTrcHisA	BamH1/EcoR1	pTrcHisA[XKS1]a	E. coli Top 10 [pTrcHisAXKS1]/– U/mg
Fw: 5′CCGGAATTCTTGTGTTC
AGTAATTCAGAGACAGACAAG3′
Rev: 5′TTCTGCAGTTAGATGAG
AGTCTTTTCCAGTTCGCT3′
Annealing temperature: 59 °C (30 cycles)	pTrc99A	EcoR1/Pst1	pTrc99A[XKS1]b	E. coli XL1-Blue [pTrc99AXKS1]/4.5 U/mg
Fw: 5′CCGGAATTCTTGTGTTCA
GTAATTCAGAGACAGACAAG3′
Rev: 5′-TTCTGCAGTTAATGATGAT
GATGATGATGGATGAGAGTCTTTTC
CAG TTCGCT-3′
Annealing temperature: 62 °C (30 cycles)	pTrc99A	EcoR1/Pst1	pTrc99A[XKS1His]c	E. coli XL1-Blue pTrc99A[XKS1His]/3 U/mg
Fw: 5′-CCGGAATTCTTGTGTTCAG
TAATTCAGAGACAGACAAG3′
Rev: 5′-TTCTGCAGTTATTTTTCGA
ACTGCGGGTGGCTCCAAGCGCTG
ATGAGAGTCTTTTCCAGTTCGCT3′
Annealing temperature: 65 °C (30 cycles)	pTrc99A	EcoR1/Pst1	pTrc99A[XKS1Strep]d	E. coli XL1-Blue pTrc99A[XKS1Strep]/2.3 U/mg
Fw: 5′-CGCGGATCCTTGTGTTCAG
TAATTCAGAGACAGACAAG-3′
Rev: 5′-CGGGAATTCTTAGATGAGA
GTCTTTTCCAGTTCGCTTAAG-3′
Annealing temperature: 59 °C (30 cycles)	pGEX-6P-2	BamH1/EcoR1	pGEX[XKS1]e	E. coli BL21 (DE3) pGEX[XKS1]/1.7 U/mg
Fw: 5′-ATACATATGTGTTCAGTAATTC
AGAGA-3′
Rev: 5′-GAAGGATCCTTAATGATGATGA
TGATGGATGAGAGTCTTTTCCAGTTCGCT-3′
Annealing temperature: 60 °C (30 cycles)	pET26b(+)	NdeI/BamHI	pET[XKS1ΔL-His]f	E. coli BL21 (DE3) pET26b(+)[XKS1ΔL] 5.6 U/mg
Fw: 5′ATACATATGGTTTGTTCA
GTAATTCAGAGA-3′
Rev: 5′GAAGGATCCTTAATGATGATGAT
GATGGATGAGAGTCTTTTCCAGTTCGCT-3′
Annealing temperature: 62 °C (30 cycles)	pET26b(+)	NdeI/BamHI	pET[XKS1V-His]g	E. coli BL21 (DE3) pET26b(+)[XKS1V-His] 3.4 U/mg

^aN-terminal His-Tag.

^bNative XKS1.

^cC-terminal His-Tag.

^dC-terminal Strep-Tag.

^eN-terminal GST-Tag.

^fLeu1 deletion mutant; C-terminal His-Tag.

^gMutant, where Leu1 was replaced by Val; C-terminal His-Tag.