Wako Pure Chemical Industries, Ltd.

Transdirect insect cell is a cell-free protein synthesis system prepared from insect cells.
Transdirect insect cell

Features

1. Transdirect insect cell is a translation system for mRNA templates.
2. Insect cell extract is prepared from Spodoptera frugiperda 21 (Sf21) insect cells, which are also widely used in the baculovirus expression system.
3. The preparation method of insect cell extract and the concentration of the reaction components are optimized to increase protein synthesis efficiency (patent pending).
4. Expression vector pTD1 (patent pending) includes the translational enhancer sequence (the 5' untranslated region derived from Malacosma neustria nucleopolyhedrovirus polyhedrin gene) suited to a cell-free protein synthesis system prepared from insect cells.

Kit components

5x Insect Cell Extract
1x Reaction Buffer
1x 4mM Methionine
1x 0.5ug/ul Control DNA
1x 0.5ug/ul pTD1 Vector

Download manual (pdf)

Appilcation data

Expression profiles of 73 human proteins

cDNA clones were obtained from KAZUSA DNA Research Institute. After centrifugation, 5uL of the supernatants were subjected to SDS-PAGE. Expressed proteins were detected by FluoroTect GreenLys (Promega).

Lane	Protein	M.W.	Lane	Protein	M.W.
1	Thioredoxin type domain	46970	38	Sec23/Sec24	121110
2	Nucleosome assembly protein	45125	39	Ubiquitin thiolesterase	127521
3	Cell division/GTP binding	49813	40	Ankyrin	123080
4	Phosphatase	55600	41	Fibronectin	125819
5	Multi TM	55015	42	C2 domain	131898
6	S-adenosyl-L-homocysteine hydrolase	67585	43	tubulin-specific chaperone	132443
7	cell cycle progression	58441	44	Ig/MHC	135193
8	Low density lipoprotein-receptor	60810	45	Tubulin-tyrosine ligase	133375
9	Sulfotransferase	64925	46	PAS	132669
10	Initiation factor eIF-4 gamma	67585	47	CPSF A subunit C-terminal	135575
11	Endoplasmic reticulum targeting sequence	72923	48	Elongation factor	138752
12	Glycoside hydrolase	73766	49	Peptidylprolyl isomerase	133618
13	Fibronectin	74329	50	Helicase	140470
14	Protein-arginine deiminase	75562	51	SH3/SAM	136679
15	Phosphatidylinositol-4-phosphate 5-kinase	73258	52	Actin-binding WH2	135614
16	Glutamines	73460	53	Myosin head	148711
17	Protein kinase	80461	54	Membrane attack complex	143986
18	Ras small GTPase	82625	55	CD2-binding protein	152396
19	Ubiquitin carboxyl-terminal hydrolase	80360	56	Multi TM	151149
20	TatD-related deoxyribonuclease	85037	57	Domain in components of the proteasome	166567
21	Guanine-nucleotide dissociation stimulator CDC25	86700	58	phosphatase	162157
22	Pyridoxal-dependent decarboxylase	86705	59	FHA	161435
23	Cold-shock DNA-binding domain	88883	60	Neurexin	164425
24	HECT domain	94221	61	BRCT domain	170675
25	SEC7-like domain	94912	62	Cell surface receptor IPT/TIG	165761
26	PAS	95302	63	Intermediate filament protein	172825
27	Multi TM	102543	64	Adipokinetic hormone	180944
28	Importin-beta	108193	65	RNP	177541
29	Meprin/TRAF-like MATH	107904	66	Mitochondrial substrate carrier	189383
30	Ubiquitin-associated domain	107411	67	Histone H1/H5	199821
31	Ribosomal protein S2	111206	68	Rap/ranGAP	199964
32	Pleckstrin-like	112778	69	Dicer1	218678
33	DEAD/DEAH box helicase	119261	70	DNA topoisomerase II	226036
34	Serine/threonine dehydratase	120413	71	S-II	229477
35	Pumilio/Puf RNA-binding	114002	72	TPR repeat/Site-specific DNA-methyltransferase	246348
36	Band 4.1 domain	120675	73	AKAP	256716
37	jumonji	121895

Productivitiy

Translational enhancer sequence

Transdirect -- baculovirus polyhedrin 5'UTR
Rabbit Reticulocyte -- beta-globin 5'UTR

Model protein: beta-galactosidase (116kDa)

(A) beta-galactosidase were synthesized in Transdirect and two commercial available rabbit reticulocyte cell-free systems. Synthesized proteins were detected by fluorescent labeled lysine after SDS-PAGE. (B) Enzymatic activities were determined by measuring degradation product of o-nitrophenyl-beta-D-galactopyranoside.

The productivity of Transdirect is approximately 10 fold higher than that of Rabbit system.

Solubility

Expression examples of mammalian proteins in Transdirect and wheat germ cell-free system.

Two mammalian proteins were expressed in Transdirect and wheat germ cell-free system. Synthesized proteins were detected by western analysis with using specific antibodies to each protein.

Solubility of expressed proteins in Transdirect are better than that in wheat germ system.

References

Cell-free protein synthesis system prepared from insect cells by freeze-thawing
Ezure, T., Suzuki, T., Higashide, S., Shintani, E., Endo, K., Kobayashi, S., Shikata, M., Ito, M., Tanimizu, K., Nishimura, O. Biotechnol. Prog., 2006, 22, 1570-1577

Performance of expression vector, pTD1, for insect cell-free translation system
Suzuki, T., Ito, M., Ezure, T., Kobayashi, S., Shikata, M., Tanimizu, K., Nishimura, O. J. Biosci. Bioeng., 2006, 102, 69-71

N-terminal protein modifications in an insect cell-free protein synthesis system and their identification by mass spectrometry
Suzuki, T., Ito, M., Ezure, T., Shikata, M., Ando, E., Utsumi, T., Tsunasawa, S., Nishimura, O.Proteomics, 2006, 6, 4486-4495