A novel flow cytometry-based method for analysis of expression levels in Escherichia coli, giving information about precipitated and soluble protein.
A high throughput method for screening of protein expression is described. By using a flow cytometer, levels of both soluble and precipitated protein can simultaneously be assessed in vivo. Protein fragments were fused to the N-terminus of enhanced GFP and the cell samples were analysed using a flow cytometer. Data concerning whole cell fluorescence and light scattering was collected. The whole cell fluorescence is probing intracellular concentrations of soluble fusion proteins. Concurrently, forward scattered light gives data about inclusion body formation, valuable information in process optimisation. To evaluate the method, the cells were disrupted, separated into soluble and non-soluble fractions and analysed by gel electrophoresis. A clear correlation between fluorescence and soluble target protein was shown. Interestingly, the distribution of the cells regarding forward scatter (standard deviation) correlates with the amount of inclusion bodies formed. Finally, the newly developed method was used to evaluate two different purification tags, His(6) and Z(basic), and their effect on the expression pattern.
Hedhammar
My
M
Royal Institute of Technology, AlbaNova University Center, Department of Biotechnology, SE-106 91 Stockholm, Sweden.
Stenvall
Maria
M
Lönneborg
Rosa
R
Nord
Olof
O
Sjölin
Olle
O
Brismar
Hjalmar
H
Uhlén
Mathias
M
Ottosson
Jenny
J
Hober
Sophia
S
D011232:Chemical Precipitation; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics* / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D015870:Gene Expression; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012995:Solubility
D011993:Recombinant Fusion Proteins