Cell Free Protein Expression

This survey is intended for scientists that have worked on cell free protein expression and are willing to share their views with us.

Please start with the survey now by clicking on the Next button below.

Which of the following describes best the company/institution you work in?

Pharmaceutical or Biotech company

Contract Research Organization or Contract Manufacturing Organization

Academia

Government

Other

What type of kits do you primarily use?

Which kind of proteins are you primarily working on?

For the work where you apply cell-free systems, why do you apply such systems rather than expressing in living cells (please choose all that apply)?

It speeds up my process

It is the fastest way to perform my high-throughput screening

My proteins do not express in living cells

I want to incorporate unnatural amino acids or other non-conventional components

To minimize purification of the protein before application (less background proteins in cell-free systems)

Other, please elaborate

Are you responsible for choosing which brand of cell-free expression kits are purchased?

Yes, I decide which cell-free expression kits are purchased in my lab in all cases

Yes, I decide which cell-free expression kits are purchased in my lab in some cases

What type of expressions do you primarily run?

Coupled batch

Uncoupled batch

Coupled continuous flow

Uncoupled continuous flow

What size cell-free reactions do you primarily perform?

How many cell-free reactions do you estimate your group perform per year?

From which companies do you primarily purchase cell lysate, i.e. cell free protein expression kits (please choose all that apply)?

Which would you prefer?

Paying 1/2 price for the kit you are accustomed to, and continue to obtain the same yield (mg/ml) of proteins

Paying the same price for the kit you are accustomed to, but obtain two-fold yield (mg/ml) of the protein

Please put in order which of the following four options would have the strongest effect on you to change the kits you use to another brand (1 having the strongest effect and 4 having the lowest effect):

Ease of scaling up my reactions from µl volumes to ml volumes
Amount of protein per ml
Reaction time
Price

How do you obtain the DNA used in the very first tep when you intend to produce a protein in a cell free system?

DNA isolated from a cell or virus

Available DNA library

DNA segment synthesized from a sequence obtained from literature, a database or similar

Other, please elaborate

What is the most frequent method you apply when preparing the DNA you add into your cell-free reaction mixture?

You PCR the DNA from your respective template with primers, which contain ribosomal binding sites and add

You PCR the DNA from a vector that contains ribosomal binding sites and add

You splice the DNA into the vector accompanying the kit and multiply in E. Coli before adding

You prepare the applicable vector from a stored frozen batch of vectors or bacteria and add

Other, please elaborate

In batch moode, the quantity of reference proteins per ml (e.g. firefly and GFP) are generally up to 1 mg/ml in prokaryotic cell-free systems and 0,2 mg/ml in eukaryotic systems. Which amount of reference protein would a new eukaryotic system have to yield for you to want to test it (please assume the price is the same as the system you are primarily using)?

What is the primary reason you would not be willing to change the kit you use to another brand?

Do you purify your protein from the lysate after it is expressed?

I don´t purify the protein before using it

I purify the protein somewhat before using it (e.g. one purification step with a tag)

I purify the protein significantly before using it (e.g. above 90% purity)

Does your protein need to move freely in the reaction mixture for you to be able to apply it, or can it be covalently bound to a membrane?

Must be able to move freely

Can be covalently bound to a membrane

What amount of protein per ml would a cell-free system kit need to yield for you to consider using such a kit for small scale production of your protein for repeated experiments and anaylsis (preparative amounts). Please assume the cost of the kit to be the same as the cost of the kit you primarily use?

0,5mg/ml

1mg/ml

2mg/ml

3mg/ml

4mg/ml

5mg/ml or more

I do not need preparative amounts of the protein(s) I work on

I am already using cell-free system to obtain preparative amounts of my protein

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