Hongyul Yoon - New Way of Isolation of Proteins
Hongyul Yoon - New Way of Isolation of Proteins
Hongyul Yoon
TRIZ Specialist, CEO of TRIZ Center, Korea
[email protected]
www.trizcenter.co.kr/eng/
ABSTRACT
It was required to extract the bindings of Prp19p and other proteins from a lipid droplet for research of
obesity repression. Extraction of some specific bindings could be done through dissociation of the other
protein bindings. SDS was adopted as a dissociating agent but it also dissociated Prp19p bindings. That
contradiction was surmounted with application of ARIZ-85C, part 1. The solution idea was proved to be
useful to lift out Prp19p bindings out of a lipid droplet and sufficiently effective for the following further
research.
1) Background
The case of this paper followed the earlier biochemical research[1]. The project of this case was
started just after Siyoung Cho and her colleagues found that Prp19p is important for further research of
repression of obesity and finished at the end of 2006 before the publication of the earlier research. For
the readers, the brief explanation of the earlier work is given below.
Repression of obesity is the main target of pharmaceutical industry for business in these days. Amore
Pacific, one of the leading companies in that area of Korea, has found that research on lipid droplet
must be the gate to conquer obesity.
Lipid droplets are subcellular organelles considered as a major energy depot by storing neutral lipids.
Therefore, the biogenesis of a lipid droplet is central to whole body energy homeostasis. Besides,
lipid droplets seem to have important roles in lipid trafficking in adipocytes, cell signaling and several
important human diseases. It is thus important to understand the mechanism of deposition and
mobilization of cellular neutral lipids. It is expected that a set of proteins involved in lipid droplet
biogenesis could be composed of effective targets for the regulation of the whole body energy
homeostasis. Lipid droplets are surrounded by a phospholipid monolayer into which many proteins are
embedded. Some of these droplet-associated proteins are reported to play important roles in the
functions of the lipid droplets. As one of them, Prp19p is an integral component of the heteromeric
protein complex(the NineTeen Complex, or NTC) in nucleus and it is essential for the structural
integrity of NTC and its subsequent activation of the splicesome. Researchers identified Prp19p as a
member of proteins associated with lipid droplets. Downregulation of Prp19p expression with RNA
interference in 3T3-L1 cells repressed lipid droplet formation with the reduction in the level of
expression of perilipin and S3-12. Prp19p or Prp19-interacting proteins during droplet biogenesis in
adipocytes may be considered as another class of potential targets for attacking obesity and obesityrelated problem.
In order to research how to control Prop19p-interacting proteins, it has to be possible to isolate the
bindings of Prp19p and other proteins. Isolation of target proteins requires dissociation of the bindings
of the other proteins in an adipocyte. In general, the recommendable dissociating agent is Sodium
Dodecyl Sulfate(SDS).
This paper is related to the problem that there was a big trouble to use SDS as a dissociating agent.
This paper says about TRIZ consultation from 15th of September to 17th of November, 2006. The
result of this case was proved to be effective and then has been used for further research.
lots of time and very often embarrasses researchers. The researchers wanted more speedy and effective
procedures for solutions. TRIZ was introduced not as the one and only way to solve the problem
situation but as a more effective way to overcome the trouble. The management of Amore Pacific R&D
Center intended to check the effectiveness of TRIZ through the project of this problem.
As shown in Fig.1, several agents like Phosphate Buffered Saline (PBS), Nonidet P 40 (NP 40), etc
are used for the process. During reaction with SDS phase seemed to cause the main problem. SDS
could be effective to separate all protein bindings except the bindings with Prp19P. However, if we
adopt SDS to the amount enough to dissociate the other cell components, the target protein combination,
or Prp19p-Proteins like Prp19p combined with soluble N-ethylmaleimide-sensitive fusion (NSF)
protein attachment protein(SNAP)-ap, might be also dissociated to be useless samples for our research.
We needed the combination of Prop19p and other not-identified proteins.
For a more clarified image of the problem, we moved on to Function Analysis of the problematic
phase.
According to the result of function analysis, we got the main problem list.
(1) harmful function problem : the function to dissociate the binding of Prp19p & proteins(in Fig. 2,
we specified the proteins as SNAPap) delivered by SDS
(2) insufficient/excessive useful function problem : there is no such a problem when the concentration
of SDS is over 4 %.
(3) contradiction problem : the physical contradiction of the concentration of SDS is related to the
technical contradiction between the dissociation of the bindings of other proteins and the undamaged
bindings of Prp19p.
6) Contradiction
Even though we have little information on the state of the inside of the dissociated cell, we could
formulate the initial contradiction for ARIZ-85C application.
ENV model of OTSM-TRIZ. Element was SDS, Name of feature was concentration, and Value of
feature was higher than 4% v.s much lower than 4%. If we had been able to solve this contradiction
through separation in time, space, and system scale, the ENV expression must have been very helpful.
ARIZ-85C 1.4
We faced the limitation of time for this project. case with diluted SDS meant that we should develop
new ways to dissociate proteins, which usually requires too much time for experiments. Researchers as
field experts couldnt follow the solving line of case with diluted SDS.
The situation made us select case with concentrated SDS at ARIZ-85C 1.4 even though the original
advice of Altshuller must have recommended case with diluted SDS.
ARIZ-85C 1.5
ARIZ-85C 1.5 produced the extreme case of what we selected ; SDS concentrated extremely higher
than 4% and complete damaged Prp19p bindings.
ARIZ-85C 1.6
- The conflicting pair ; SDS concentrated extremely higher than 4% as tool,
Prp19p bindings as product
- The intensified formulation of the conflict ; SDS concentrated extremely higher than 4% dissociates
the other protein bindings in a lipid droplet but it also dissociates the target bindings of Prp19p.
- It is necessary to find an X-element, which would prevent dissociation of the target bindings of
Prp19p, while keeping the property of the extremely concentrated SDS to dissociate the other protein
bindings.
ARIZ-85C 1.7
ARIZ-85C 1.7 was run, which asks us to apply inventive standards of Altshuller to our exaggerated
problem model. In the exaggerated condition, we are satisfied with the dissociation function of SDS.
The problem is dissociation of Prp19p bindings. For application of inventive standards, we interpreted
the problem by substance-field model;
S1 : Prp19p bindings
S2 : SDS at very high concentration (greatly over 4%)
interaction between S1 and S2 : harmful action
Through this mapping thinking from the function model or ENV model to a substance-field model, we
find several recommendations of rule 1-2-1~5. Rule 1-2-1 prompted an idea shown in Fig. 5.
9) Conclusion
Through OTSM-TRIZ approach, we got the world-first effective way to isolate Prp19p-combined
proteins from lipid droplets of a human cell. ENV model and multi-screen thinking based on OTSMTRIZ gave us the systematic thinking way to transform the non-typical problem into a kind of typical
problem. Through ARIZ-85C part 1, we got the idea as a potential solution after a shorter period and less
trials than usual. The developed world-first way to take out the target protein Prp19 bindings has been
used for the next step of obesity repression research since the end of 2006.
REFERENCES
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Song, I.S. Chang, O.S.Lee, T.R.Lee, Identification of mouse Prp19p as a lipid droplet-associated protein
and its possible involvement in the biogenesis of lipid droplets, J. Biol. Chem. 282(2007) 2456-2465.
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