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Transcript
The Use of Cell-Free Systems to Produce Proteins for
Functional and Structural Applications
Julia Fletcher, Shiranthi Keppetipola, Ashley Getbehead, Federico Katzen and Wieslaw Kudlicki
Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, California 92008 • USA
Figure 1 – More functional protein
Results
Figure 3 – 100% incorporation of
Selenomethionine into a
membrane protein
Figure 5 – Cell-free expression of
SUMO for NMR analysis
Se‐Met
188
98
62
98
62
49
49
38
28
•Reactions are easily scalable and flexible for expression of desired amounts of protein without the need for any special equipment. 38
EmrE
EmrE
28
17
14
17
14
6
6
M L FT W E1 E2 E3 M L FT W E1 E2 E3 T FT W1 W2 E1 E2 E3 E4 M M
15N labeled‐
Sumo
15
Feed
Reaction
Incubate
30 min
1 ml
Continue incubation up to 6 hrs
Milligram
quantities of
protein
1 ml
Figure 2 – Mg amounts of product
2 ml
100
90
80
70
60
50
40
30
20
10
0
13600
14299.03 EmrE Methionine 2480.1
14486.58 EmrE Seleno‐
methionine
Figure 6 – N-HSQC spectra from crude
and purified preparations of SUMO
Purified
•The Expressway™ Milligram system can be used to efficiently incorporate Selenomethinone for crystal structure analysis.
•Expressway™‐NMR minimizes the amounts of labeled amino acids (1ml feed vs. 10ml dialysis) required for labeling due to smaller reaction volumes.
•Expressway™‐NMR is supplied with a separate amino acid module allowing desired concentrations to be tailored as needed.
14200
14800
•Protein expression and labeling in the Expressway™‐NMR system provides protein samples sufficient for NMR analysis without the need for purification.
0
15400
Mass (m/z)
Invitrogen’s Expressway™ Milligram and NMR systems use a patented buffer formulation of novel compounds to regenerate the diphospho‐ and monophospho‐nucleotides and maintain a pool of available NTPs for in vitro protein synthesis. These intermediates are supplied to the reaction through discrete “feeds”, in a very simple format, requiring no special equipment or devices to generate milligram levels of protein. These intermediates work in conjunction with an enhanced E. coli extract to drive higher yields of functional synthesized proteins (Figures 1 and 2). A single one‐milliliter reaction combined with a one‐milliliter feed can generate milligrams of soluble, biologically active protein. Reduced levels of amino acid scrambling make it possible to efficiently label the products with modified amino acids. The products are suitable for protein crystallization and NMR spectroscopy (Figures 3, 4, 5, and 6). The addition of a mix of detergents and liposomes to the reaction permitted, for the first time, the synthesis of milligram levels of an integral membrane protein suitable for crystallization (Figures 3 and 4). Finally, the Expressway™ NMR protein expression system provides protein samples of adequate concentration and purity for direct NMR analysis (Figure 6).
Conclusions
• Expressway™‐Milligram generates milligram quantities of proteins in a single reaction simply through the addition of feeding buffer during the reaction.
kDa
Met
%Intensity
Abstract
Robust and dependable protein expression systems are the subject of increasing demands in the areas of genomics and proteomics, regardless whether studying single or multiple proteins from a functional or structural standpoint. Invitrogen’s
Expressway™ systems allow the in vitro synthesis of recombinant proteins in a single reaction tube or in a multiplexed format in a just a few hours. We now report two additions to this product line, which are targeted to the structural proteomics field: Expressway™ Milligram and Expressway™ NMR. The core of these two kits consists of an improved buffer system and cell lysate that allows the synthesis of milligram amounts of protein in a 2‐ml‐
reaction format in just 3 hours using circular or linear DNA templates. The Expressway™ Milligram has been used to produce milligram amounts of otherwise difficult to express proteins such as membrane proteins. Functional and structural data will be presented. Expressway™ NMR provides an efficient and economical platform for stable‐isotope labeling of proteins. By employing this kit, protein purification is not longer needed to obtain clean and reliable HSQC‐spectra in under five hours.
Figure 4 – Crystals and Diffraction
pattern of a membrane protein
synthesized in vitro
References
Crude
Crystals of Selenomethionine‐labeled EmrE
Diffraction from crystals (up to 3.8A)
Pornillos, O.,Chen, Y. J.,Chen, A. P. and Chang, G., 2005. X‐ray structure of the EmrE multidrug transporter in complex with a substrate. Science. 310, 1950‐3.
Keppetipola, S., Kudlicki, W., Nguyen, B. D., Meng, X ., Donovan, K. and Shaka
AJ., 2005. From Gene to HSQC in Under Five Hours: High Throughput NMR Proteomics. JACS (submitted) [In collaboration with Scripps Research Institute (G Chang laboratory, Pornillos et al, 2005), Invitrogen’s Research Grant]
[In collaboration with the AJ Shaka laboratory, UC Irvine]
Expressway.Milligram Cell‐Free E. coli Expression System with pEXP5‐NT/TOPO®
and pEXP5‐NT/TOPO® ,5 rxns, Cat No. K9900‐96 without vector, 5 rxns K9900‐97
Expressway.NMR Cell‐Free E. coli Expression Systemwith pEXP5‐NT/TOPO® and pEXP5‐
NT/TOPO®, 5 rxns, Cat No. K9900‐98, without vector, 5 rxns, Cat No. K9900‐99
Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, California 92008 USA • Telephone: 760 603 7200 • FAX: 760 602 6500 • Toll Free Telephone: 800 955 6288 • E-mail: [email protected] • www.invitrogen.com