This VNp_Cargo_yield readme.txt file was generated on 2022/01/17 by Dan Mulvhill

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GENERAL INFORMATION
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1. Title of Dataset 
Exceptional yield vesicular recombinant protein production from E. coli.

2. Author Information

  Principal Investigator Contact Information
        Name: Daniel P. Mulvihill
           Institution: University of Kent
           Address: School of Biosciences, Canterbury, Kent, CT2 7NJ, UK
           Email: d.p.mulvihill@kent.ac.uk

  Associate or Co-investigator Contact Information
        Name: Tara A. Eastwood
           Institution: University of Kent
           Address: School of Biosciences, Canterbury, Kent, CT2 7NJ, UK
           Email: T.A.Eastwood@kent.ac.uk

 Associate or Co-investigator Contact Information
        Name: Karen B. Baker
           Institution: University of Kent
           Address: School of Biosciences, Canterbury, Kent, CT2 7NJ, UK
           Email: K.B.Baker@kent.ac.uk

3. Date of data collection: 20210201 to 20220113

4. Geographic location of data collection (where was data collected?):
School of Biosciences, University of Kent, Canterbury. CT2 7NJ

5. Information about funding sources that supported the collection of the data:
BBSRC - BB/S005544/1

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DATA & FILE OVERVIEW
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Filename: Gel Densitometry Analyses of VNp fusion proteins     
Short description:  pdf file containing scanned stained SDS-PAGE gels on which soluble cell extracts or cleared media samples have been run. Defined amounts of BSA were run on each gel for contraction calibration during densitometry analysis.
These gels were used to determine yields of recombinant proteins in the associated paper.

             

3. Additional related data collected that was not included in the current data package:

4. Are there multiple versions of the dataset? no

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METHODOLOGICAL INFORMATION
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1. Description of methods used for collection/generation of data: 
Bacterial cell culture and protein induction: All bacterial cells were cultured at 37 ºC in either LB (10 g Tryptone; 10g NaCl; 5g Yeast Extract (per litre)) or TB (12g Tryptone; 24g Yeast Extract; 4ml 10% glycerol; 17 mM KH2PO4 72 mM K2HPO4 (per litre)) media. 5 ml LB starters from fresh bacterial transformations were cultured at 37 ºC to saturation and used to inoculate 25 - 500 ml volume TB media, flask cultures that were incubated overnight at 37 ºC with 200 rpm orbital shaking. Recombinant protein expression from the T7 promoter was induced by addition of IPTG to a final concentration of 20 µg / ml (except Etanercept and hGH where 10 µg / ml was used) once the culture had reached an OD600 of 0.8 – 1.0). To generate amino-terminally acetylated VNp, target constructs were co-transformed into E. coli with pNatB, to allow co-expression with the fission yeast amino--acetyltransferase complex B, Naa20 and Naa25 1. Amino-terminal acetylation was confirmed by electrospray mass spectroscopy of the purified VNp fusion protein. Growth curves were generated from 96 well plate cultures, prepared from late log-phase cultures, diluted into fresh media to an OD600 of 0.1 at the start of the growth analysis experiment. OD600 absorbance values were obtained using a Thermo Scientific Multiscan Go 1510-0318C plate reader and recorded using the SkanIt Software 4.0. at OD600 values were taken every 30 minutes for the duration of the experiment, and growth curves generated from averages of ≥ 3 individual biological repeats. 
Soluble protein extracts: Cell pellets from 25ml of a 50 ml culture were resuspended in 5 ml of soluble extract buffer (20 mM TRIS, 500 mM NaCl, pH 8.0), sonicated for a total of 2 min (6 x 20 sec pulses), and cell debris removed by centrifugation at 39,000 xg (4 ºC) for 20 min. Target protein concentration was determined using fluorescence of mNeonGreen fusion or gel densitometry. Both techniques were compared directly on the same samples to determine equivalence.
Recombinant vesicle isolation: Vesicles were isolated directly from overnight bacterial cell cultures by passing the culture through a sterile and detergent-free 0.45 µm polyethersulfone (PES) filter. 

2. Methods for processing the data: <describe how the submitted data were generated from the raw or collected data>
Protein concentration determination: Fluorescence scanning was used to determine concentration of mNeongreen fusion proteins in vesicle containing media and soluble protein extracts. Absorbance was measured at 506 nm using a Varian Cary 50 Bio UV-Vis spectrophotometer, with measurements from conditioned media used for baseline correction, and concentration determined using an extinction coefficient of 116,000 M-1cm-1. Concentration of non-mNeongreen labelled proteins was determined by gel densitometry analysis of triplicate samples run alongside BSA loading standards on coomassie stained SDS-PAGE gels.

Determination of cytosolic VNp concentration: VNp-mNeongreen expression was induced in BL21 DE3 E. coli for 4 hours at 37ºC (when extracellular vesicle production is observed), and images of more than 120 cells were acquired using widefield imaging (described below) from 3 independent sample preparations. Mean mNeongreen intensity was determine from a 5 x 5 pixel area within each E. coli cell. A calibration line was generated from multiple images of known concentrations of VNp-mNeongreen solutions using identical imaging conditions as cell image acquisition. 

3. Instrument- or software-specific information needed to interpret the data:
See above.

4. Standards and calibration information, if appropriate:
See above.

5. Environmental/experimental conditions:
See above.

6. Describe any quality-assurance procedures performed on the data:
Average yields were calculated (mg soluble target protein / litre culture) from a minimum of 3 independent biological repeats from cultures of BL21 DE3 E. coli cells grown in TB media.

7. People involved with sample collection, processing, analysis and/or submission:
Data acquisition and analysis: T. A. Eastwood and K. B. Baker
Submission: D. P. Mulvihill

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DATA-SPECIFIC INFORMATION FOR: [Gel Densitometry Analyses of VNp fusion proteins]
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<create sections for each dataset (or file if appropriate) included>

1. Number of variables: N/A

2. Number of cases/rows: N/A

3. Variable List 
    A. Name: [variable name]
       Description: [description of the variable]
                    Value labels if appropriate

    B. Name: [variable name]
       Description: [description of the variable]
                    Value labels if appropriate

4. Missing data codes: N/A
        Code/symbol        Definition
        Code/symbol        Definition

5. Specialized formats of other abbreviations used:
VNp: Vesicle Nucleating Peptide.
mNG: mNeongreen
LZ: Leucine Zipper
hGH: Human Growth Hormone
EPO: Erythropoietin
FGF: Fibroblast Growth Factor
DARP: Designed Ankyrin Repeat Protein