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ARIZ-85 to Isolate Target Proteins

Editor

By Hongyul Yoon

Abstract

It was required to extract the bindings of Prp19p and other proteins from a lipid droplet for obesity repression research. Extraction of some specific bindings could be done through dissociation of the other protein bindings. Sodium dodecyl sulfate was adopted as a dissociating agent but it also dissociated Prp19p bindings. That contradiction was surmounted with the application of ARIZ-85C, Part 1. The solution idea was proved to be useful to lift Prp19p bindings out of a lipid droplet and sufficiently effective to research.

Background

The case of this paper followed some earlier biochemical research.1 This case started just after Siyoung Cho and her colleagues found that Prp19p was important for the research of the repression of obesity. The research finished at the end of 2006 before the publication of the earlier research; a brief explanation of the earlier work follows.

The repression of obesity is a main target in the pharmaceutical industry. Amore Pacific, one of the leading companies in that area of Korea, found that lipid droplets were the way to conquer obesity.

Lipid droplets are sub-cellular organelles that are a major energy depot; they store neutral lipids. The biogenesis of a lipid droplet is central to the whole body energy homeostasis. Lipid droplets also seem to have important roles in lipid trafficking in adipocytes, cell signaling and several 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 hospholipid 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 the 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. Down regulation 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 obesity-related problems.

In order to research how to control Prp19p-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 recommended dissociating agent is sodium dodecyl sulfate (SDS). This paper is related to the problem that there was trouble using SDS as a dissociating agent. This paper reflects a Theory of Inventive Problem Solving (TRIZ) consultation from September 15-November 17, 2006. The result of this case was proven effective and has been used for further research.

Why TRIZ Was Introduced

It is probable that the researchers would have been searching for different dissociating agents as an alternative to SDS through trial-and-error, if TRIZ had not been introduced. Trial-and-error requires lots of time. The researchers wanted faster and more effective procedures for finding solutions. TRIZ was introduced not as the only way to solve the problem, but as a more effective way to overcome the problem. The management of Amore Pacific R&D Center intended to check the effectiveness of TRIZ throughout this project.

Initial Problem Situation: Problem Identification

Just like the general solving process of a certain problem, the first step of this case was the identification of the problem. It is not likely that a team willidentify the ideal final result (IFR) at the start of a project. Generally, the IFR can not be confirmed until the correct contradiction or function relationship is formulated. For correct formulation of contradictions and function relationship, several preliminary steps were needed.

The first step is identifying what we want. It was effective for problem solving to re-identify what was desired with the ENV (Element – name of feature – value of feature) model of OTSM-TRIZ.2,3This model helps to abstract the essence of the problem, which is unlikely to be revealed in a short sentence.

ENV results from feedback along the problem solving process. After ENV identification of “what we want,” it is time to move to the next step. These steps are the author’s recommendation for real TRIZ application. Table 1 shows the final ENV model of this case.

Table 1: ENV Model to Identify the Project’s Goal

Value of Feature

Element

Name of Feature

Current

Desired

Other cell components

Position

With Prp19p+ proteins

Awayfrom Prp19p+ proteins

Through the ENV model of the goal the general ways to isolate the target, “Prp19p-combined protein,”could be abstracted. Actually, there was no information about what proteins combine with Prp19p in a lipid droplet and it was necessary to dissociate cell components by SDS. SDS was the only detergent that had been found that could complete the required dissociation to all cell components. This situation led to the selection of “co-immunoprecipitation with SDS” as the way to separate the target protein bindings.

Multi-screen Thinking Based on OTSM-TRIZ

With OTSM-TRIZ, the phase of the problem was identified through multi-screen thinking.2,4Multi-screen thinking based on OTSM-TRIZ enabled the identification of which phase should be addressed among several phases of the whole co-immunoprecipitation process.

As shown in Table 2, several agents like phosphate buffered saline (PBS) and Nonidet P 40 (NP 40) 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 SDS is changed to the amount needed 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 also be dissociated to be useless samples for this research. The combination of Prp19p and other not-identified proteins was needed. For a more clarified image of the problem, it was necessary to move on to the function analysis stage of the problematic phase.

Table 2: Problem Phase Identification According to Multi-screen Thinking Based on TRIZ

Environmental Components

Time -2: Before Introduction of SDS

Time -1: During Reaction with SDS

Time 0: SDS Introduction and Mixing

Time 1: Detecting (Anti-body Introduction)

Super-system

Other protein, HO NP40, sodium deoxy cholate, PBS

Other protein, H2O NP40, sodium deoxy cholate, PBS

Other protein, H2O NP40, sodium deoxy cholate, PBS

Anti-body: 2 types

System

Prp19p+SNAPap+lipid droplet: binded

?

Prp19p, SNAPap, lipid droplet (all of them are dissociated)

Sub-system

Prp19p, SNAPap, lipid droplet

?

Prp19p, SNAPap, lipid droplet

^^Function Analysis ^^

Function Analysis

The function analysis was done for the identified problem phase.4The result of the function analysis would be used for Part 1, Step 2 of ARIZ-85C. Function analysis on chemical/biochemical systems should be supported by the interpretation of the chemical interactions in terms of physical changes.5The researchers tried to identify the interactions among entities in a cell when SDS was introduced. They had no technically precise information about the real phenomena – the model was built focusing only on the target entities and relying on previous knowledge and imagination. This model could not be using when analyzing the phenomenon in a cell; however, this model could be a good reference. The goal of function analysis is not a solution idea list but a problem list for applying TRIZ thinking ways to the problem situation.

Figure1 shows the function analysis of “during reaction with SDS” when the concentration of SDS is higher than 4 percent. SDS with a concentration higher than 4 percent is enough to dissociate all bindings among proteins.

According to the result of function analysis, the main problem list was developed:

  1. Harmful function problem: the function “to dissociate the binding of Prp19p and proteins (in Figure 1, the proteins were specified as SNAPap) delivered by SDS”
  2. Insufficient/excessive useful function problem: there is no such a problem when the concentration of SDS is greater than 4 percent.
  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.
Figure 1: Function Analysis of the
Identified Problem Phase

Contradiction

Even though the researchers have little information on the state of the inside of the dissociated cell, the initial contradiction for ARIZ-85C application can be formulated.

Figure 2: Key ContradictionFrom the Function Analysis

For a clearer description of the problem situation, the physical contradiction was formulated in an ENV model of OTSM-TRIZ. The element was SDS, name of feature was concentration, and the value of feature was higher than 4 percent vs. much lower than 4 percent. If they had been able to solve this contradiction through a separation in time, space and system scale, the ENV expression would have been helpful.

Application of ARIZ-85C, Part 1

If the researchers had enough information about the problematic phenomenon, they might have applied ARIZ-85C to the contradiction obtained from the function analysis. Biochemical/chemical problems usually seem to accompany a shortage of knowledge of the problem situation, often resulting in a case with which only a kind of discovery could show how to solve the problem.

Based on experiences problem solving with OTSM-TRIZ, the researcher found that only Part 1 of ARIZ-85C could reduce the searching area for problem solving, or achieve “the key task of problem solving” in OTSM-TRIZ terms.6 The author ran ARIZ Part 1 as a tacit procedure for problem solving. It is not necessary to run ARIZ explicitly for its application. If the consultant is familiar with ARIZ, it is enough to guide the team members along the process of ARIZ.

ARIZ-85C 1.1

This step was done based on the contradiction presented in Figure 2. The system for the isolation of Prp19p bindings includes SDS, Prp19p and the other proteins in a lipid droplet.

  • Technical contradiction 1: If a SDS concentration higher than 4 percent is applied, the other protein bindings are completely dissociated but the target protein bindings with Prp19p also are dissociated.
  • Technical contradiction 2: If an SDS concentrationdiluted much lower than 4 percent is applied, the target protein bindings with Prp19p are kept undamaged but the other protein bindings are not completely dissociated.

It is necessary to dissociate the other protein bindings completely without damaging the Prp19p bindings by introducing minimal change.

ARIZ-85C 1.2

ARIZ-85C 1.2 resulted from the model of function analysis: tool is SDS, product is Prp19p bindings and other parts of the lipid droplet.

ARIZ-85C 1.3

The models required by ARIZ-85C 1.3 are shown in Figure 3.

Figure 3: Diagram for ARIZ-85C 1.3 Acquired From Function Analysis

ARIZ-85C 1.4

The researchers faced the limitation of time for this project. “Case with diluted SDS” meant that new ways to dissociate proteins needed to be developed, which usually requires too much time for experiments. Researchers as field experts could not follow the solving line of “case with diluted SDS.” The situation led to the selection of “case with concentrated SDS” with ARIZ-85C 1.4 even though the original advice of TRIZ father Genrich Altshuller and his colleagues must have recommended “case with diluted SDS.”

ARIZ-85C 1.5

ARIZ-85C 1.5 produced the extreme case of what the researchers selected; an SDS concentration significantly higher than 4 percent and complete damage to the Prp19p bindings.

ARIZ-85C 1.6

  • The conflicting pair: SDS concentration significantly higher than 4 percent as tool, Prp19p bindings as product.
  • The intensified formulation of the conflict: SDS concentrated significantly higher than 4 percent 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 the 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, in which the researchers applied the inventive standards to the exaggerated problem model. In the exaggerated condition, the researchers are satisfied with the dissociation function of SDS. The problem is the dissociation of Prp19p bindings. To apply the inventive standards, the researchers interpreted the problem by a substance-field model approach.

S1: Prp19p bindings
S2: SDS at extremely high concentration (significantly greater than 4 percent) interaction between S1 and S2: harmful action

Through this mapping, the thinking from the function model or ENV model to a substance-field model, the researchers found several recommendations of rule 1-2-1~5. Rule 1-2-1 prompted an idea shown in Figure 4.

Figure 4: IdeaFrom ARIZ-85C 1.7

This idea suggests the introduction of an intermediate blocker between SDS and the target protein combination.

Solution Ideas and Result

Based on the basic idea of ARIZ-85C 1.7, the researchers selected several potential intermediate blocking agents among several candidates below:

  • 3,3´-Dithiobis [sulfosuccinimidylpropionate] (DTSSP)
  • 4-succinimidyloxycarbonyl-α-methyl-α-[2-pyridyldithio]toluene (SMPT)
  • Dimethyl 3,3’-dithiobispropionimidate•2HCl (DTBP)
  • Etc.

The test results in Figure5 say that DSP (dithiobis succinimidyl propionate) would be the best cross-linking agent for this case. Figure6 shows that the proteins through this new process successfully respond to provide the required information for further research on how to control the proteins combined with Prp19p in lipid droplets.

Figure 5: Test Results for Potential Cross-linking Agents
Figure 6: Gel
Test Result

Conclusion

Through the combination of OTSM-TRIZ, the researchers found an effective way to isolate Prp19p-combined proteins from the lipid droplets of a human cell. The use of the ENV model and multi-screen thinking (based on OTSM-TRIZ) provided a path of systematic thinking to transform the non-typical problem into a typical type of problem. Through ARIZ-85C Part 1, the solution idea developed with a shorter time period and fewer trials than usual. The developed first-in-the-world 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.

Acknowledgement

Siyoung Cho, Taeryong Lee, Daegun Kim, Byungfhy Suh from the R&D Center, Amore Pacific Corporation, Korea.

References

  1. Cho, S.Y.,E.S. Shin, P.J. Park, D.W. Shin, H.K. Chang, D.K. Kim, H.H. Lee, J.H. Lee, S.H. Kim M.J. Song, I.S. Chang, O.S. Lee, T.R.L ee, “Identification of Mouse Prp19p as a Lipid Droplet-associated Protein and its Possible Involvement in the Biogenesis of Lipid Droplets,” Journal ofBiological Chemistry, Vol. 282, Issue 4, 2456-2465, January 26, 2007.
  2. Khomenko, Nikolai,OTSM-TRIZ, Training Hand-outs, LG Electronics, 2000.
  3. Khomenko, Nikolai, Comments on new problem technology of OTSM-TRIZ, 2002-2007.
  4. Ikovenko, Sergei, Lecture materials on TRIZ, LG Chem, 2003.
  5. Yoon, Hongyul, TRIZ lecture materials for chemical industry, Amore Pacific, 2006.
  6. Yoon, Hongyul, Advanced course materials on modern TRIZ tools, RIST, 2006.
  7. Altshuller, Genrich, Creativity as an Exact Science, Gordon and Breach Science Publishers, New York, 1988.
  8. Salamatov, Yuri, TRIZ: The Right Solution at the Right Time, INSYTEC B. V., 1999.