An efficient purification method for high recovery of ...

1 International Journal of Environmental Science and Development, Vol. 1, No. 2, June 2010. ISSN:2010-0264. An efficient purification method for high recovery of Recombinant Human Granulocyte Colony Stimulating Factor from recombinant E. coli 1+. S. Abolghasemi Dehaghani ,V. Babaeipour 2+, M. R. Mofid 3, A. Divsalar 4,F. Faraji 1. Abstract Human G-CSF, a single chain polypeptide I. INTRODUCTION. containing 174 amino acid residues (MW=18,800, pI= ), is one Development of inexpensive and simple culture media is of the hemopoietic growth factors. Development of inexpensive and simple culture media is always favorable for commercial always favorable for commercial production of recombinant production of recombinant proteins in E.

2 Coli. proteins in E. coli. Many of the efforts aimed at increasing The high -level expression of eukaryotic proteins in E. coli recombinant protein production in bacterial strains have been often leads to formation of insoluble inclusion bodies (IBs) in the directed to maximizing the biomass production with the high cytoplasm or periplasm. recovery of active material from (IBs) cell density cultivation method and little is known about the is often difficult and involves two general steps: protein effects of media composition on the expression of solubilization in a denaturant and protein refolding.

3 Recombinant proteins [1, 2]. On a commercial scale, reducing the number of protein purification steps is practical and economical because each Hence In this study, at first the effects of medium purification step not only increases the final product but also composition on the production of recombinant human causes successive yield losses of the recombinant protein. Granulocyte-Colony Stimulating Factor (rh-GCSF) was In this research, we developed an efficient and scalable investigated in batch culture; and then a simple and procedure for production and purification of recombinant cost-effective downstream process for the economical human (rh-GCSF) of E.

4 Coli. This process include: an optimized production of rh-GCSF is developed. batch culture with LB and glucose 10 g/l with expression level 40%, cell harvesting, cell lyses with high pressure homogenizer, The high -level expression of eukaryotic proteins in E. coli two steps washing, IB solubilization, refolding, and finally often leads to formation of insoluble inclusion bodies (IBs) in protein purified by FPLC with cation exchanger column. By the the cytoplasm or periplasm. Inclusion bodies are dense but using of the new developed method , of g l-1 rh-GCSF was porous particles of aggregated protein that Usually only one produced in each batch, 720 mg pure of recombinant protein or a few different proteins are inside the IB, and no ribosomal was obtained with recovery yield about 40% and purity over components or nucleic acids are resent and they are held than 99%.

5 According to available data this is one of the highest together by non-covalent hydrophobic or ionic bonds. yield and production level of the purified recombinant protein that has been reported for human recombinant protein which is IBs have some native-like protein structures, but also have expressed in E. coli. Also with this procedure we can produce a an increased amount of non-native -sheet [3]. Three Factors protein that the structural characterization was preserved. that contribute to inclusion body (IB) formation: 1) Inclusion bodies form most often as a result of Index Terms rh-GCSF; Escherichia coli; Inclusion bodies; overexpression of a non-native protein.

6 purification ; Solubilization; Refolding 2) Hydrophobic proteins form IBs more readily. 3) Non-native proteins with disulfide bonds are prone to form IBs because the disulfide bridge cannot form in the cytosol Accidental oxidation can also lead to improper S. Abolghasemi Dehaghani is with the Dept. Biology Science & disulfide formation[ 3,5]. Research Branch Islamic Azad University, Tehran, Iran, (corresponding Culture conditions such as temperature, pH, and nutrient author to provide phone:; fax: +98+21-88970258; e-mail: ). supply play a very important role in controlling the partition V. Babaeipour is with the Biochemical Engineering Group, of the recombinant protein into soluble and insoluble Biotechnology Research Center, Tehran, Iran,(corresponding author to fractions.

7 recovery of active material from (IBs) is often provide phone:; fax: +98+21-22974605; e-mail: difficult and involves two general steps: protein Mofid is with the Isfahan University of medical sciences (e-mail: mofid solubilization in a denaturant and protein refolding [3]. In A. Divsalar is with the Department of Biological Sciences, Tarbiat general, proteins expressed as inclusion bodies are Moallem University, Tehran, Iran,( e-mail: ) solubilized by the use of high concentrations of chaotropic F. Faraji is with the Dept. Biology Science & Research Branch Islamic Azad University, Tehran, Iran,( e-mail: solvents Human G-CSF, a single chain polypeptide containing 174.))

8 111. International Journal of Environmental Science and Development, Vol. 1, No. 2, June 2010. ISSN:2010-0264. amino acid residues (MW=18,800, pI= ), is one of the performed twice. hemopoietic growth factors which plays an important role in D. Rh-GCSF purification stimulating proliferation, differentiation, and functional activation of blood cells. It contains a free cysteine at position Cell lysis and IB recovery : The fermented broth was 17 and two intramolecular disulfide bonds [4]. centrifuged at 4 C and 8000 g for 30 min and the obtained When rh-GCSF is produced by E. coli, the formation of pellet was washed twice with 50 mM phosphate buffer pH.

9 Disulfide bonds is either incorrect or inhibited because the reducing environment of bacterial cytosol, and it is The wet cells (50 g) were suspended in 200 ml of lysis accumulated in the form of IBs. buffer. The lysis buffer composition was 50 mM Tris HCl Earlier reports indicate that the levels of rh-GCSF containing 1 mM EDTA, 1 mM PMSF. The cells were expressed in E. coli was at moderate to high levels (10 35%) broken by passing the medium through a homogenizer three [7, 8], and the yield of the final product was very poor and far times (NIRO-SOAVI) at 800 bar. The cells were cooled to from acceptable.

10 This is probably because of unproductive 4 C between each pass. The cell homogenate was centrifuged downstream process technologies like isolation of protein for 30 min at 6000g at 4 C, the supernatant was discarded IBs with low purity and recovery , misfolding, aggregate and the inclusion bodies recovered. formation and unoptimized conditions of protein refolding, IBs washing: The IBs pellet obtained in previous step was and chromatographic processes [5]. Therefore in this resuspended in wash buffer and incubated 40 min and research has been tried to develop a well-organized and recovered by centrifugation at 25 28 C for 30 min at 8000 g.