Name: cell-free protein expression using the rapidly growing bacterium vibrio natriegens
Uploaded: 2019-03-14T15:00:00
Duration: 8 min 52 s
Description: Watch this Scientific Journal Video about Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens at JoVE.com

This work was funded by the National Institute of General Medical Sciences 1U01GM110714-01 and Department of Energy DE-FG02-02ER63445. The authors would like to thank Dr. Richard Kohman, Dr. Jenny Tam, and Dr. Edgar Goluch for helpful advice on constructing the protocol section of this manuscript.

<ol>
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DJW, NO, and GMC have filed a patent related to this work.

This protocol has been optimized for wild-type V. natriegens and bacterial growth media comprised of LB supplemented with V2 salts (Table of Materials). Other strains of V. natriegens can be similarly cultured to generate crude cell extracts for cell-free reactions; however, their use requires additional optimization of this protocol. Additionally, this cell-free protein expression system has been optimized using 3-phosphoglyceric acid (3-PGA) as the primary energy regeneration source. Other energy regeneration sources may be used; however, optimization of reagents and calibration will likely be required to obtain high yielding protein expression10,11.
Specific attention to several critical steps in this protocol will ensure maximal extract productivity for high-yielding cell-free protein production. First, crude cell extract must be prepared from V. natriegens cell cultures harvested in a mid-exponential phase of growth; protein yield is maximal when cultures reach an OD600 = 1.0 &#177; 0.2. While cell-free protein production is possible from cells harvested at a range of optical densities, we have previously found that cells harvested in an exponential state of growth yield significantly more protein9. The observed effects of optical density on crude cell extract performance are consistent with those reported for other cell-free expression systems derived from cells grown in batch culture conditions1. Because V. natriegens grows at a rapid rate, it is critical to closely monitor cultures&#8217; optical densities. In general, it is expected that V. natriegens cultures should reproducibly reach an OD600 of 1.0 within 1&#8722;1.5 h using this protocol; however, individual growth conditions such as the use of baffled versus non-baffled flasks, which affects aeration, or air versus water incubation, which affects the rate and stability of incubation temperature, may alter growth time. Furthermore, it is generally recommended to culture at least 250 mL of V. natriegens in a 1 L baffled flask to ensure a large cell pellet at harvest for easy manipulation and transfer. This greatly improves the success of crude cell extract preparation as well as increases the total volume of extract produced from a pellet. When using smaller scale preparation, culture conditions and reagents can be adjusted appropriately. For large-scale fermentation, further optimization of culture conditions may be required. Finally, to ensure high protein yield, it is critical that cell pellets are processed immediately after harvesting, or within 1&#8722;2 days of storing at -80 &#176;C.
The proper lysis of the cell pellet by pulse sonication is critical to the success of cell-free protein expression and is often the most difficult aspect of this protocol for new users. Typically, a well lysed pellet will yield a significant volume of liquid extract that is free of debris. The extract should be slightly viscous but can be easily pipetted when aliquoting into storage tubes before flash freezing in liquid nitrogen. Figure 3 depicts a representation of a well lysed pellet (Figure 3A) in comparison to a poorly lysed pellet (Figure 3B) after the post lysis centrifugation step. A major indication of complete cell lysis is a crude cell extract total protein concentration &#62; 20 mg/mL as determined by a total protein assay (step 2.13). Over-sonication or excessive heating of the crude cell extract will damage the cellular machinery, which cannot be determined without performing a cell-free reaction. Thus, it is highly beneficial to test extract efficiency with a control reaction before dedicating significant time and effort to downstream protein expression applications. While additional optimization may be necessary for different sonication equipment, the pulse sonication steps described have been highly reproducible in our hands.
The use of linear DNA template derived from PCR amplification, restriction enzyme digest, or commercial gene synthesis can significantly increase the capacity for high-throughput and rapid protein production in cell-free expression systems24. While protein production from PCR amplified linear template has been demonstrated, the yield of these reactions are approximately 13.5-fold lower compared to reactions using plasmid DNA template at equimolar ratios9. This is primarily due to the instability of the linear DNA template which is likely degraded by endogenous nucleases present in V. natriegens crude cell extract. While lambda phage protein GamS has been previously used to protect Linear DNA template24,25, it was found to be incompatible with V. natriegens extracts9. Additionally, while increasing the concentration of linear DNA template may allow for a higher protein yield, its fast degradation in crude cell extract will still be a major problem.
A solution to overcoming linear DNA template degradation may be to supplement cell-free reactions with mRNA template generated from in vitro transcription of linear DNA. On the other hand, the use of an RNase inhibitor offers significant protection against mRNA transcript degradation and appreciable protein yields can be obtained in the 10 &#956;L cell-free reaction format (Figure 5A,B). Cloning of linear DNA into a circular template through TA ligation, TOPO cloning, Golden Gate assembly, or other recombination methods may be used to circumvent template degradation. Nevertheless, further approaches for inhibition of nuclease activity will be necessary for efficient protein expression using linear DNA template.
To date, several different approaches have been proposed for preparation of crude extract for cell-free protein expression9,10,11,26. In developing this protocol, we sought to maximize user accessibility, reduce overall cost, and minimize time-consuming steps. For example, a high protein yield is achieved using a simple two-step sonication-centrifugation process, and does not require cell homogenizers, lengthy dialysis steps or run-off reaction. It is simple to execute in a short period of time and does not require high level of laboratory expertise. Thus, it can help facilitate cell-free expression as a standard for translational academic research and industrial process design.
This protocol expands the toolkit available for investigation and utility of V. natriegens, a non-model organism with unique biological properties. Higher protein yield can be achieved by employing semi- or fully-continuous cell-free reactions, to allow for energy regeneration, resupplying of amino acids, and the removal of waste products3,5,27. Furthermore, the engineering of wild-type V. natriegens to produce DNAse- or RNAse-deficient strains, removal of deleterious and competing metabolic pathways, and expression of additional tRNAs could greatly enhance the production of proteins in this system28,29. As we unravel the biology underlying its rapid growth, further development of V. natriegens cell-free systems may accelerate bioproduction capabilities and enable robust expression of therapeutic peptides, small molecules, and synthetic materials.

Cell-free protein synthesis is a versatile and cost-effective method for the expression of valuable proteins or peptides1,2,3,4. Historically, cell-free protein synthesis has been performed using Escherichia coli expression systems; however, there has been a recent surge in using alternative, non-model organisms with novel properties as chassis for cell-free expression5,6,7,8,9,10,11,12. Organisms with unique metabolic profiles are prime candidates as alternatives to E. coli cell-free systems. For example, the marine bacterium Vibrio natriegens is the fastest growing of all known organisms with an observed doubling time of less than 10 min13. This has garnered V. natriegens considerable attention as an emerging microbial host for research and biotechnology14,15,16,17,18. Given that the rapid growth rate of V. natriegens has been linked to high rates of protein synthesis and metabolic efficiency19,20,21, harnessing its cellular machinery for cell-free synthesis may significantly expand the toolkit for rapid protein production and high-throughput screening.
Recently, a cell-free V. natriegens expression system has been demonstrated which is capable of producing super folder GFP (sfGFP) at concentrations of &#62; 260 &#956;g/mL in 3 h with a T7 promoter9. The overall aim of developing this method was to provide users with a highly accessible, cost-efficient, reproducible, and high-yielding cell-free protein expression system that can be prepared using common lab equipment in a short amount of time. This protocol utilizes 1 L cultures in shake flasks, cell lysis by pulse sonication, and small-scale batch reactions in a 96- or 384-well format to maximize parallelization and screening throughput. A long, sustained protein expression is made possible by supplementation of 3-phosphoglyceric acid (3-PGA) as an energy source8,22,23. Upon successfully completing this protocol, a user will have the capability to express a desired protein or set of proteins in a cell-free format using V. natriegens crude cell extract.
Starting from a glycerol stock, V. natriegens crude cell extracts are prepared from cells harvested at an optical density at 600 nm (OD600) of 1.0. A 1 L culture will yield approximately 2&#8722;3 mL of extract, which is sufficient for more than 800 cell-free reactions at 25% crude cell extract. Proteins can be expressed using plasmid DNA, linear DNA, or mRNA template; however, linear DNA template degradation by endogenous nucleases remains a major drawback when using wild type V. natriegens cell-free system9. Starting from V. natriegens cultures, protein useable for downstream applications can be achieved by a single user in 1&#8722;2 full days.

<table>
	<tbody>
		<tr>
			<td>15 mL Tubes</td>
			<td>Corning</td>
			<td>352196</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Tubes</td>
			<td>Eppendorf</td>
			<td>22363352</td>
			<td></td>
		</tr>
		<tr>
			<td>384-well Black Assay Plates</td>
			<td>Corning</td>
			<td>3544</td>
			<td></td>
		</tr>
		<tr>
			<td>384-well PCR Plates</td>
			<td>Eppendorf</td>
			<td>951020702</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Tubes</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well PCR Plates</td>
			<td>Eppendorf</td>
			<td>30129300</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 3&#39;,5&#39;-cyclic monophosphate sodium salt monohydrate</td>
			<td>Sigma</td>
			<td>A6885</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#39;-triphosphate - 100 mM</td>
			<td>NEB</td>
			<td>N0450L</td>
			<td></td>
		</tr>
		<tr>
			<td>Applied Biosystems Veriti 384-well Thermocycler</td>
			<td>ThermoFisher</td>
			<td>4388444</td>
			<td></td>
		</tr>
		<tr>
			<td>Assay Plate Adhesives</td>
			<td>BioRad</td>
			<td>MSB1001</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Nicotinamide adenine dinucleotide hydrate (NAD)</td>
			<td>Sigma/Roche</td>
			<td>10127965001</td>
			<td></td>
		</tr>
		<tr>
			<td>Coenzyme A hydrate</td>
			<td>Sigma</td>
			<td>C4283</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytidine 5&#39;-triphosphate - 100 mM</td>
			<td>NEB</td>
			<td>N0450L</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(-)-3-Phosphoglyceric acid disodium salt (3-PGA)</td>
			<td>Sigma</td>
			<td>P8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Dewar Flask - 4L</td>
			<td>ThermoScientific</td>
			<td>10-194-100C</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol solution - 1 M</td>
			<td>Sigma</td>
			<td>42816</td>
			<td></td>
		</tr>
		<tr>
			<td>Folinic acid calcium salt hydrate</td>
			<td>Sigma</td>
			<td>47612</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Sigma</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanosine 5&#39;-triphosphate - 100 mM</td>
			<td>NEB</td>
			<td>N0450L</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamic acid hemimagnesium salt tetrahydrate</td>
			<td>Sigma</td>
			<td>49605</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamic acid potassium salt monohydrate</td>
			<td>Sigma</td>
			<td>G1149</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth (Miller)</td>
			<td>Sigma</td>
			<td>L3522</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2)</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid pJL1-sfGFP</td>
			<td>Addgene</td>
			<td>69496</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid Plus Maxi kit</td>
			<td>Qiagen</td>
			<td>12963</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(ethylene glycol) (PEG)-8000</td>
			<td>Sigma</td>
			<td>89510</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hydroxide Pellets</td>
			<td>Sigma/Roche</td>
			<td>1050121000</td>
			<td></td>
		</tr>
		<tr>
			<td>Q125 Sonicator and CL-18 probe with a &#8539;-inch tip</td>
			<td>Qsonica</td>
			<td>4422</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Clean and Concentrator Kit</td>
			<td>Zymo</td>
			<td>R1013</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Inhibitor, Murine</td>
			<td>NEB</td>
			<td>M0314</td>
			<td></td>
		</tr>
		<tr>
			<td>RTS Amino Acid Sampler</td>
			<td>Biotechrabbit</td>
			<td>BR1401801</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma</td>
			<td>S0266</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 RNA Polymerase</td>
			<td>NEB</td>
			<td>M0251</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Solution (pH 8.0) - 1 M</td>
			<td>Invitrogen</td>
			<td>AM9856</td>
			<td></td>
		</tr>
		<tr>
			<td>tRNA from E. coli MRE 600</td>
			<td>Sigma/Roche</td>
			<td>10109541001</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine 5&#39;-triphosphate - 100 mM</td>
			<td>NEB</td>
			<td>N0450L</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrio natriegens (Wild-type) Lyophilized Stock</td>
			<td>ATCC</td>
			<td>14048</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell-free protein expression using the rapidly growing bacterium vibrio natriegens

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A general protocol is described for the preparation of V. natriegens crude cell extracts using common laboratory equipment. This high yielding protocol has been specifically optimized for user accessibility and reduced cost. Cell-free protein synthesis (CFPS) can be carried out in small scale 10 &#956;L batch reactions in either a 96- or 384-well format and reproducibly yields concentrations of &#62; 260 &#956;g/mL super folder GFP (sfGFP) within 3 h. Overall, crude cell extract preparation and CFPS can be achieved in 1&#8722;2 full days by a single user. This protocol can be easily integrated into existing protein synthesis pipelines to facilitate advances in bio-production and synthetic biology applications.

Watch this Scientific Journal Video about Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens at JoVE.com

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

Cell-free expression systems are powerful and cost-efficient tools for the high-throughput synthesis and screening of important proteins. Here, we describe the preparation of cell-free protein expression system using Vibrio natriegens for the rapid protein production using plasmid DNA, linear DNA, and mRNA template.

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A ...

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