Hi Itisam, have you sequenced your constructs? Are they ok? (any accidentally introduced stops, mutations, signal sequences, cleavage sites etc. ?)
Are the "lots of glycines" you mention just glycines (worst case: e.g. GGG GGG GGG GGG GGG GGG - just guanines) or do you have some variation in the codons? This can cause horrible frameshifts. A linker that is used frequently is eg GSSGSSGSSG or something alike, this will be more hydrophilic than just glycines. Have you considered codon usage (frequency) to avoid limited expression by limiting (rare) tRNAs? You may check eg the "codon usage database" (use google or whatever to find it). Does your construct work without GFP? Everything there what you need for successful expression, like Kozak consensus before the Start, appropriate promoters etc? If everything within your construct is ok, there is some chance that your vector has been damaged during propagation / cloning. -> Try subcloning your construct into a backbone (or just another clone with the same backbone) that is known to work. Consider having the complete construct "chemically" synthesized. If you consider time and materials, this might be by far the cheapest and quickest option meanwhile. You may get codon usage optimization included for free. And you have someone to blame when it doesn't work :) If you like, check this example for tethering a lysosomal protein to the outer surface of mammalian cells: http://www.ncbi.nlm.nih.gov/pubmed/19101472 HTH Wo Am Tue, 20 Dec 2011 13:49:45 +0800 schrieb Itisam Sarangi: > Hi, > I have one problem. it might be a becoz of some fundamental problem. I > want to express some cytosolic proteins (1000 amino acids) on cell > surface. so i can use these for my expts. > > Construct design : > N-terminus flag tag, a signal peptide ( from ICAM molecule) followed > by a > cloning site and a trans-membrane domain with a short tail (from ICAM) > and 2A- GFP. > > there are lot of glycines in between to help for proper folding. i use > 10+ amino acids more on either side of trans-membrane domain. > > > problems: > 1.Without the signal peptide but with trans-membrane domain, i see flag > tag on surface of GFP +ve cells but with signal peptide no flag tag > staining. which is understandable as signal is chopped off along with > flag tag. hence no staining. > > 2. when expressed the gene of intererst, with out signal but with > membrane i donot see the expression of protein in gfp +ve cells. > > > The probbable reasons > -> proteins are toxic hence degraded. -> signal peptide not strong to > guide protein to membrane. any other strong signal peptide or change in > position of signal peptide in construct. > > If any one has any experince/ suggestions are welcome > > > thanks _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
