Hello!.
I have to express a protein in S. cerevisiae with pYES2 vector.
This protein has a molecular weight of 56 kDa and a His tag for the purpose of purification with ProBond resin.
In the process of purifying the protein, the purified protein has a molecular weight of 30 kDa.
Which are the possible sources of error in my poor expression protein of interest?
Hi there,
First :Do you observe any full length protein before purification?
Second: The position of the histag is important in order to try to understand what is going on. As you manage to purify some protein it means the histag is present. So if at the Nter, it means either the full length protein is degraded/cleaved at Cter or there is a stop codon within the cloned ORF. If at the Cter, you might have Nter degradation/cleavage only. Other thing to do : try to identify the purified protein sequence (Nter sequencing, MS).
Hi there,
First :Do you observe any full length protein before purification?
Second: The position of the histag is important in order to try to understand what is going on. As you manage to purify some protein it means the histag is present. So if at the Nter, it means either the full length protein is degraded/cleaved at Cter or there is a stop codon within the cloned ORF. If at the Cter, you might have Nter degradation/cleavage only. Other thing to do : try to identify the purified protein sequence (Nter sequencing, MS).
You should look into the codon usage adaptation index. Maybe your sequence have rare codons or with low frequency. You can check here: http://www.genscript.com/cgi-bin/tools/rare_codon_analysis
Correlation between Protein and mRNA Abundance inYeast
Steven P. Gygi, Yvan Rochon, [...], and Ruedi Aebersold
ABSTRACT
We have determined the relationship between mRNA and protein expression levels for selected genes expressed in the yeast Saccharomyces cerevisiaegrowing at mid-log phase. The proteins contained in total yeast cell lysate were separated by high-resolution two-dimensional (2D) gel electrophoresis. Over 150 protein spots were excised and identified by capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein spots were quantified by metabolic labeling and scintillation counting. Corresponding mRNA levels were calculated from serial analysis of gene expression (SAGE) frequency tables (V. E. Velculescu, L. Zhang, W. Zhou, J. Vogelstein, M. A. Basrai, D. E. Bassett, Jr., P. Hieter, B. Vogelstein, and K. W. Kinzler, Cell 88:243–251, 1997). We found that the correlation between mRNA and protein levels was insufficient to predict protein expression levels from quantitative mRNA data. Indeed, for some genes, while the mRNA levels were of the same value the protein levels varied by more than 20-fold. Conversely, invariant steady-state levels of certain proteins were observed with respective mRNA transcript levels that varied by as much as 30-fold. Another interesting observation is that codon bias is not a predictor of either protein or mRNA levels. Our results clearly delineate the technical boundaries of current approaches for quantitative analysis of protein expression and reveal that simple deduction from mRNA transcript analysis is insufficient.
The description of the state of a biological sys
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC83965/
use double tags when u are cloning.. both C & NTR!
like eg His and Strep... Pichia has huge problem of protease mediate digestion.. this could be the case may... use PMSF resuspension to lysis..
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