In situ cell-free expression of unknown proteins

Progres Meeting Madrid, 2012

1616 Genes on Chromosome 16

B-lymphocyte T-lymphocyte Leukocyte Epithelium Fibroblast

Entries 538 244 263 491 329

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Protein coding genes (n=862)

Entr

ies

More than 85 % of protein coding genes of chromosome 16 are expressed in lymphoid cells, epitelial cells and fibroblasts.

1616 Genes on Chromosome 16

B-lymphocyte T-lymphocyte Leukocyte Epithelium Fibroblast

Entries 538 244 263 491 329

50

150

250

350

450

550

Protein coding genes (n=862)

Entr

ies

More than 85 % of protein coding genes of chromosome 16 are expressed in lymphoid cells, epitelial cells and fibroblasts.

Mix two plasmids and enzymeOnly the Desired Product Survives

Master

Moving Genes by Recombination

Gateway Technology for cloning & subcloning

•Master Library Many libraries•Any vector can be modified to accept genes

•Master Library Many libraries•Any vector can be modified to accept genes

Testing Proteins Many Different Ways

Testing Single Proteins in Many Different Ways

Where is the protein

located in the cell?

Produce lots of

protein for experiment

s.

Turn the protein on and off in mammalian

cells.

Test the protein’s function in

mammalian cells.

What other proteins does this protein

interact with?

Perform assays using protein made in

bacteria.

Marcador 1Kb LRRC29 MC1R

KCNG4

TMEM170

GSG1LIGSF6 CKLFSF2

WFDC1

Insert Verification by Xho1 & BamHI Digestion

Detection of in situ cell-free protein expression

WB Beads Suspension Arrays (BBAs)

tagged protein ontoanti-GST beads

Cell-free expression of GST-tagged DNA

MixDetection

Anti-GST Anti-mouse

Coupling of anti-GST antibody to beads

Capture of GST-

re-aliquot

Beads Suspension Arrays