Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protein Science > Recombinant Protein Forum
Register Search Today's Posts Mark Forums Read

Recombinant Protein Forum Recombinant Protein Forum. Discuss purification, expression, and isolation of recombinant and cloned proteins here.


Using Chromatography to perform protein folding of recombinant proteins

Using Chromatography to perform protein folding of recombinant proteins - Recombinant Protein Forum

Using Chromatography to perform protein folding of recombinant proteins - Recombinant Protein Forum. Discuss purification, expression, and isolation of recombinant and cloned proteins here.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 10-27-2009, 08:20 PM
danfive's Avatar
Nobel Laureate
Points: 8,612, Level: 64 Points: 8,612, Level: 64 Points: 8,612, Level: 64
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2007
Location: Houston TX
Posts: 969
Thanks: 12
Thanked 184 Times in 154 Posts
Post Using Chromatography to perform protein folding of recombinant proteins



Here's a review article covering the use of all kinds of chromatography for recombinant protein refolding, to recover native state and of course bioactivity. Very useful, helped me figure out why my size-exclusion columns produced nil; apparently, these methods can be a real time saver, 20 min chromatography can be equivalent to 24 hours of dialysis.

Protein folding liquid chromatography and its recent developments
Xindu Geng∗, Chaozhan Wang


---> [Only registered users see links. ]

Introduction
One of the purposes of proteomics is to identify unknown
biologically active proteins and use this information to develop
novel drugs. Some active proteins occur at very low levels in the
human body and thus have to be produced by biotechnology.
Escherichia coli is one of the mostly used host cell in biotechnology.
But when proteins are expressed in E. coli, they often
form inactive protein aggregates called inclusion bodies. A step
necessary in recovering active proteins from E. coli is protein
refolding (it is simply called protein folding here) or protein
renaturation; it is usually the key step during the production of
therapeutic proteins by biotechnology, especially at the industrial
scale. The yields from refolding by traditional methods are
usually very low, typically 5–20%. Application of liquid chromatography
(LC) to protein folding is one of the most interesting
and exciting methods to develop in recent years. When it is used
in protein folding, the bioactivity recovery increases, the folded
protein can be easily separated from misfolded forms, protein
concentration after refolding is relatively high, and it is easy to
scale up and automate, therefore it is regarded as an efficient,
and close to ideal refolding method [1,2]. Additionally, it has
the potential to be used at an industrial or large scale, today it
has become a very popular technique for protein folding.

Protein refolding by liquid chromatography can be simply
named as “protein folding liquid chromatography”. It is
defined as “a kind of liquid chromatography, with various kinds
of biochemical and/or physicochemical processes originally
accomplished in solution, which can result in either raising the
efficiency, or shortening the time of protein folding” [3].

An ideal PFLC should have the following four functions
depicted simultaneously in Fig. 1 [3]. They are the removal
of denaturants, refolding of target proteins, separation from
contaminant proteins including misfolded intermediates of the
target protein, and easy recovery of denaturants. It usually takes
20–40 min to complete a chromatographic run with simultaneous
protein folding. In addition, by continuously changing
the components of the mobile phase, different proteins can be
separated with suitable folding conditions to refold and simultaneously
purify in only one chromatographic run.

By using the normal dilution method for protein folding,
denaturants and contaminant proteins cannot be removed. Some
precipitates of target proteins will form during dilution; this not
only results in a low recovery, but also requires centrifugation
after an overnight incubation. Therefore, the target protein must
be further processed using coarse fractionation and fine fractionation.

In addition, using the usual dialysis method for protein
folding, it typically takes 24 h to refold a protein, with numerous
changes of buffer during dialysis. This method can remove
most of the denaturants, but cannot completely remove them, and
cannot separate the target protein from contaminant proteins.

In the past years, Guo and Geng [4], Li et al. [5], Jungbauer et
al. [6], Middelberg [2] and two books [3,7] separately introduced
PFLC and reviewed its development from different aspects. A
comprehensive review of this field is presented in this paper,
including their principles, recent developments and applications,
apparatus, recent developments for PFLC at large scale, and
effecting factors were summarized and discussed.
Reply With Quote
The Following User Says Thank You to danfive For This Useful Post:
Muhammad Rahman (12-11-2009)
  #2  
Old 04-04-2010, 04:23 PM
Pipette Filler
Points: 871, Level: 16 Points: 871, Level: 16 Points: 871, Level: 16
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Mar 2010
Posts: 10
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Using Chromatography to perform protein folding of recombinant proteins

Thanks so much! I spent an inordinate amount of time trying to produce soluble protein because I wanted to avoid all of the downstream steps associated with purifying and refolding inclusion bodies. I will definitely try to make this work.
Reply With Quote
Reply

Tags
chromatography , folding , perform , protein , protein folding , proteins , recombinant


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Human Cytome Project - Update 24 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 1 08-01-2010 02:18 PM
Human Cytome Project - an idea - Update 19 April 2005 Peter Van Osta Cell Biology and Cell Culture 1 06-01-2009 02:17 PM
A Human Cytome Project - an idea - Update 14 March 2005 Peter Van Osta Cell Biology and Cell Culture 0 03-14-2005 02:27 PM
Human Cytome Project - Update 6 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 0 01-06-2005 11:18 AM
New Saccharomyces Sequences 05/19/04 SGD Sequences Yeast Forum 0 05-23-2004 04:06 PM


All times are GMT. The time now is 04:40 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.18748 seconds with 16 queries