المرجع الالكتروني للمعلوماتية
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An overview of protein isolation  
  
1389   02:04 مساءاً   date: 13-4-2016
Author : Clive Dennison
Book or Source : A guide to protein isolation
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Date: 18-4-2016 2219
Date: 17-4-2016 1497
Date: 13-4-2016 2886

An overview of protein isolation

 

Isolating a protein may be compared to playing a game of golf.  In golf, the  player is faced with a series of problems,  each unique and yet similar to problems  previously  encountered.  In  facing  each  problem  the player must analyse the  situation and decide, from experience,  which  club is likely to  give the  best result in the given circumstances.  Similarly, in attempting to isolate proteins, researchers face a series of similar-yet-unique problems.  To solve these they must dip into their bags and select an appropriate technique.  The  purpose of this  book  is thus  to  fill  the beginnerís “golf bag”  with techniques  relevant  to  protein  isolation, hopefully to improve their game.

Developing protein isolation is also somewhat like finding a route up a mountainside.  Different routes have to be explored and base-camps established at each stage.  Occasionally it will be necessary to return to the base of the mountain for further supplies, and haul these up to the established camps, before the  next  stage  can  be attacked.  A successful climb is always rewarding and if an efficient route  is established,  it  may become a pass, opening the way to further discoveries.

 

1.1 Why do it?

This book is about the  methods that  biochemists use to  isolate proteins, and so it may  be asked,  “why  isolate  proteins?”  Looked  at  in one way, living organisms may be regarded as machines with features  in common with the  entities that we  commonly think of as “machines”.  A typical machine is made of a number of parts which interact, transduce energy, and bring about some  desired effect.  Mechanical machines have moving parts, while electronic machines move electrons. “Engines” convert energy to mechanical motion.  Internal combustion engines, for example, convert  chemical energy to mechanical motion.  Similarly, living organisms such as the human body are complex machines made up of many  interacting  systems.  Proteins  constitute  the  majority  of the working parts  of these  systems and there  are thus diverse reasons  for isolating proteins, viz.;

• To gain insight.  As with any mechanism, to  study the  way in  which a living system works it  is necessary  to  dismantle  the  machine  and to isolate the  component  parts  so that  they  may  be studied, separately and in their  interaction  with  other  parts.  The  knowledge that  is gained in this  way may  be  put  to  practical  use,  for  example,  in  the design of medicines, diagnostics, pesticides, or industrial processes. Many proteins may themselves be used as “medicines” to make up for losses or inadequate synthesis. Examples are hormones, such as insulin, which is used in the therapy  of diabetes, and blood fractions, such as the  so-called Factor  VIII, which is used in the therapy of haemophilia.  Other proteins may be used in medical diagnostics, an example being the  enzymes  glucose oxidase and peroxidase, which are used to measure glucose levels in biological fluids, such as blood and urine.

•  For use in Industry.  Many enzymes are used in industrial processes, especially where the materials being processed are of biological origin. In every case a pure protein  is desirable as impurities may either be misleading, dangerous or unproductive, respectively.  Protein isolation  is, therefore, a very common, almost central, procedure in biochemistry.

 

1.2 Properties of proteins that influence the methods used in their study

It must be appreciated  that  proteins  have  two properties  which determine the  overall  approach  to  protein  isolation  and make this different from the approach used to isolate small natural molecules.

• Proteins are labile.  As molecules go, proteins are relatively  large and delicate and their  shape  is easily changed, a process called denaturation, which leads to loss of their biological activity.  This means that  only mild procedures can  be used and techniques  such as boiling and distillation,  which are  commonly  used in  organic chemistry, are thus verboten.

• Proteins  are  similar  to one  another.  All proteins are composed of essentially the  same amino  acids and differ only  in the  proportions and sequence of their amino acids, and in the 3-D folding of the  amino acid chains.  Consequently  processes  with  a  high  discriminating potential are needed to separate proteins.

The combined requirement for delicateness yet high discrimination means that, in a word, protein separation techniques have to be very subtle. Subtlety, in fact, is required of both techniques and of experimenters in biochemistry.

 

1.3 The conceptual basis of protein isolation

In a protein  isolation one is endeavouring to  purify a particular protein, from some biological (cellular) material, or from a bio product, since proteins are only synthesized by living systems.  The  objective  is to separate the protein of interest from all non-protein material and all other proteins which occur in the same material.  Removing the other proteins is the difficult part because, as noted above, all proteins  are similar in their gross  properties.  In an ideal case, where one was able to remove the contaminating proteins, without any loss of the  protein  of interest, clearly the total amount of protein would decrease while the activity (which defines the particular protein of interest) would remain the same (Fig. 1 .).

Figure 1. A schematic representation of protein isolation.

 

Initially (Fig.  1A)  there  is a small amount  of the  desired protein  “a” and a large amount of total protein “b”. In the course of the isolation, ìbî is  reduced and ultimately  (Fig.  1B)  only  ìaî  remains,  at  which  point “a”=“b”. Ideally, the amount of “a” remains unchanged but, in practice, this is seldom achieved and less than  100% recovery of purified protein is usually obtained.

As a general principle, one should aim to achieve the isolation of a protein;-

• in as few steps as possible and,

 • in as short a time as possible.

This minimizes losses and the  generation  of isolation  artifacts.  Also,  to further study the  protein,  the  isolation  will have  to  be done many  times over and the effort put  into  devising a quick, simple,  isolation  procedure will be repaid many  times  over,  in subsequent savings. The  overall approach to the  isolation of a protein is shown in Fig.  2.

Figure 2.  An overview of protein isolation.

 

1.3.1 Where to start?

To isolate a protein,  one  must start  with some way of measuring the presence of the protein and of distinguishing it from all other  proteins that might be present in the same material.  This is achieved by a method which measures (assays) the unique activity of the protein.  With  such an assay, likely materials can be analyzed in order to select one  containing  a large amount of the protein of interest, for use as the starting material. Having selected a source material, it is necessary to  extract  the protein into  a soluble form  suitable for manipulation.  This may be achieved by homogenizing  the  material  in  a  buffer  of low  osmotic strength (the low osmotic pressure helps to lyse cells and organelles), and clarifying the extract by filtration and/or centrifugation steps.

The clarified extract is typically subjected to preparative fractionation, at this stage usually by salting out as this also usefully serves to separate protein  from  non-protein material.  It is necessary to assay the fractions obtained, in order to select the fraction(s) containing the protein of interest.  The selected fraction(s) can then be subjected to further preparative fractionation,  as required, until a pure fraction  is obtained.

Experience has shown that there  is  an optimal sequence in which preparative methods may be applied.  As a first approach  it is best to apply salting out (or TPP)  early in the  procedure, followed by ion-exchange or affinity chromatography.  Salting out can, with advantage, be followed by hydrophobic  interaction  chromatography,  because hydrophobic interactions are favoured by high salt concentration,  so desalting is obviated. The precipitate obtained from TPP, however,  is low in salt and so can be applied directly to  an ion-exchange system, without prior desalting.  Generally, molecular exclusion  chromatography should be reserved for late  in  the  isolation  when  only  a few components remain, since it is not  a highly discriminating technique.  Affinity chromatography often achieves the desirable aims of a rapid isolation using a minimum number of steps and so it should always be explored  and preferentially used where possible.

1.3.2 When to stop?

How can one know when the fraction  is pure,  i.e.  when to  stop?  To obtain this information  it  is necessary to  analyse  the  isolated fraction using a number of analytical fractionation methods.  If a number of such analytical methods reveal the apparent presence of only one protein,  it may be inferred that the protein is pure, and that the  isolation has been successfully completed. Note, however, that it is not possible to  prove that the protein is pure; one can merely fail  to  demonstrate  the  presence of impurities.  Future,  improved,  analytical  methods  may  reveal impurities that are not detected using current technology.

If, on the  other  hand,  any analytical  fractionation  method demonstrates the presence  of more  than  one  protein,  it may  be inferred that the  preparation  is not  pure.  In  this  case, the  application  of further preparative fractionation methods may be required before the protein is finally purified.

As illustrated in Fig.  1, the  requirement is to  remove  as much contaminating protein as possible, while retaining  as much as possible of the desired protein.  Clearly then, to monitor the progress of an isolation, one needs two assays, one for the activity of the protein of interest (expressed in units of activity/ml)  and another for the protein content (expressed as mg/ml).  The activity per unit of protein  (units/mg) gives a measure of the so-called specific activity.  In the course of a successful protein isolation, the specific activity should increase  with each step, reaching a maximum value when the protein is pure.  It is also desirable that  a maximum  yield of the  protein  is obtained.  The protein of interest is defined by  its  activity  and  so  information  concerning  the yield may also be obtained from activity assays.

 

1.4 The purification table

The results of activity and protein assays, from a protein purification, are typically summarized in a so  called purification  table,  of which Table 1 is an example.

Table 1. A typical enzyme purification table

 

The figures in Table  1 are arrived at as follows:-   Volume (ml) this refers to the measured total  solution volume  at the particular stage in the isolation.

•  Total protein  (mg)  -  the primary  measurement  is  of  protein concentration, i.e. mg ml-1, which is obtained using a protein assay. Multiplying the  protein  concentration  by the total  volume  gives the total protein (i.e. mg/ml x ml = mg).

•  Total activity (units)  the activity,  in units ml-1, is obtained  from  an activity assay.  Multiplying the activity by the  total  volume gives the total activity (i.e. units/ml x ml = units).

•  Specific-activity (units/mg)  -  the specific  activity is  obtained  by dividing the  total  activity  by the  total  protein.  Alternatively,  the activity (units/ml) can be divided by the protein concentration (mg/ml), in which case the miles cancel  out, leaving units/mg.

•  Purification  (fold) 

  fold refers  to  the  number  of  multiples  of  a starting value.  In  this  case  it  refers  to  the  increase  in  the  specific activity, i.e. the purification is obtained by dividing the specific activity at any stage by the specific activity  of the original homogenate.  The purification  “per step”  can also  be  obtained by dividing the specific activity after that step by the  specific activity  of the material before that step.

•  Yield (%) - the yield is based on the recovery of the activity after each

step. The activity of the original homogenate is arbitrarily set at 100%. The yield (%) is calculated from the total activity (units) at each step divided by the  total  activity  (units)  in  the  homogenate, multiplied by 100.  The  yield can  also  be calculated  on  a  “per  step” basis by dividing the total activity after that step by the  total  activity before that step and multiplying by  100.

The efficiency of a step - is calculated as:-

 

References

Dennison, C. (2002). A guide to protein isolation . School of Molecular mid Cellular Biosciences, University of Natal . Kluwer Academic Publishers new york, Boston, Dordrecht, London, Moscow 




علم الأحياء المجهرية هو العلم الذي يختص بدراسة الأحياء الدقيقة من حيث الحجم والتي لا يمكن مشاهدتها بالعين المجرَّدة. اذ يتعامل مع الأشكال المجهرية من حيث طرق تكاثرها، ووظائف أجزائها ومكوناتها المختلفة، دورها في الطبيعة، والعلاقة المفيدة أو الضارة مع الكائنات الحية - ومنها الإنسان بشكل خاص - كما يدرس استعمالات هذه الكائنات في الصناعة والعلم. وتنقسم هذه الكائنات الدقيقة إلى: بكتيريا وفيروسات وفطريات وطفيليات.



يقوم علم الأحياء الجزيئي بدراسة الأحياء على المستوى الجزيئي، لذلك فهو يتداخل مع كلا من علم الأحياء والكيمياء وبشكل خاص مع علم الكيمياء الحيوية وعلم الوراثة في عدة مناطق وتخصصات. يهتم علم الاحياء الجزيئي بدراسة مختلف العلاقات المتبادلة بين كافة الأنظمة الخلوية وبخاصة العلاقات بين الدنا (DNA) والرنا (RNA) وعملية تصنيع البروتينات إضافة إلى آليات تنظيم هذه العملية وكافة العمليات الحيوية.



علم الوراثة هو أحد فروع علوم الحياة الحديثة الذي يبحث في أسباب التشابه والاختلاف في صفات الأجيال المتعاقبة من الأفراد التي ترتبط فيما بينها بصلة عضوية معينة كما يبحث فيما يؤدي اليه تلك الأسباب من نتائج مع إعطاء تفسير للمسببات ونتائجها. وعلى هذا الأساس فإن دراسة هذا العلم تتطلب الماماً واسعاً وقاعدة راسخة عميقة في شتى مجالات علوم الحياة كعلم الخلية وعلم الهيأة وعلم الأجنة وعلم البيئة والتصنيف والزراعة والطب وعلم البكتريا.