Furthermore, sufficient expression and interaction with Rubisco activase are major challenges as well. One successful method for expression of Rubisco in E. coli involves the co-expression of multiple chloroplast chaperones, though this has only been shown for Arabidopsis thaliana Rubisco. Depletion in proteomic studies Due to its high abundance in plants (generally 40% of the total protein content), RuBisCO often impedes analysis of important signaling proteins such as transcription factors, kinases, and regulatory proteins found in lower abundance (10-100 molecules per cell) within plants. For example, using mass spectrometry on plant protein mixtures would result in multiple intense RuBisCO subunit peaks that interfere and hide those of other proteins. Recently, one efficient method for precipitating out RuBisCO involves the usage of protamine sulfate solution. Other existing methods for depleting RuBisCO and studying lower abundance proteins include fractionation techniques with calcium and phytate, gel electrophoresis with polyethylene glycol, affinity chromatography, and aggregation using DTT, though these methods are more time-consuming and less efficient when compared to protamine sulfate precipitation. Evolution of RuBisCO Phylogenetic studies The chloroplast gene rbcL, which codes for the large subunit of RuBisCO has been widely used as an appropriate locus for analysis of phylogenetics in plant taxonomy. Origin Non-carbon-fixing proteins similar to RuBisCO, termed RuBisCO-like proteins (RLPs), are also found in the wild in organisms as common as Bacillus subtilis. This bacterium has a rbcL-like protein with a 2,3-diketo-5-methylthiopentyl-1-phosphate enolase function, part of the methionine salvage pathway. Later identifications found functionally divergent examples dispersed all over bacteria and archaea, as well as transitionary enzymes performing both RLP-type enolase and RuBisCO functions. It is now believed that the current RuBisCO evolved from a dimeric RLP ancestor, acquiring its carboxylase function first before further oligomerizing and then recruiting the small subunit to form the familiar modern enzyme. The small subunit probably first evolved in anaerobic and thermophilic organisms, where it enabled RuBisCO to catalyze its reaction at higher temperatures. In addition to its effect on stabilizing catalysis, it enabled the evolution of higher specificities for over O2 by modulating the effect that substitutions within RuBisCO have on enzymatic function. Substitutions that do not have an effect without the small subunit suddenly become beneficial when it is bound. Furthermore, the small subunit enabled the accumulation of substitutions that are only tolerated in its presence. Accumulation of such substitutions leads to a strict dependence on the small subunit, which is observed