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P a g e | 11
Cloning and expression of a Synthetic Gene Coding for Mini-proinsulin in Escherichia coli
Essam. H. Ibrahim
a,b*, Tamer H. Elshaarawy
c, Abdel Jawad M. Hashem
d, Saad M Bin Dajem
a, Kalid Al Sayaad
a, Hamed A. Ghramh
a,e, Ali Alshehri
a, Mona Kilany
a,f, Eman R. Elbealy
gaBiology Department., Faculty of Science, King Khalid University, Abha, Aseer, Saudi Arabia, 61413
bBlood Products Quality Control and Research Department, National Organization for Research and Control of Biologicals, Agouza, Cairo, Egypt.
cEgyptian Holding Company for Biological Products and Vaccines (VACSERA), Agouza, Cairo, Egypt.
dDepartment of Microbiology & Immunology, Faculty of Pharmacy, Cairo University, Cairo, Egypt
eResearch Center for Advanced Materials Science (RCAMS), King Khalid University, P.O. Box 9004, Abha 61413, Saudi Arabia
Keywords: Mini-proinsulin, human insulin, Cloning and expression, Escherichia coli, Synthetic Gene.
1. INTRODUCTION
Since the beginning of recombinant DNA technology era, a very large number of recombinant proteins have been extensively produced in different host organisms (1).
However, this field is still of a very limited application in Egyptian pharmaceutical industry. Egyptian industrial organizations that are concerned with biological products still depend on imported final biotechnology products or imported recombinant raw materials for local formulation. In best cases, some organizations may depend on imported biotechnology processes through technology transfer programs. Therefore, trying to develop an expression system for the production of recombinant proteins of high value both biologically and commercially is of special interest for the Egyptian research. Human insulin represents an excellent model for therapeutic recombinant proteins with biologic and commercial high values because it is considered as the basic treatment for all type І diabetes and a several numbers of type ІІ diabetic patients. Diabetes mellitus represents a significant
public health problem as it affects a large number of the world population. The number of persons who have diabetes has been raised from 108 million in 1980 to reach 422 million by the year 2014 (2). Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and lower limb amputation. In 2012, an estimated 1.5 million deaths were directly caused by diabetes and another 2.2 million deaths were attributable to high blood glucose (3). Actually, diabetes mellitus represents a significant public health problem in Egypt and Saudi Arabia (4). In 2015, it was estimated that the prevalence of type 2 diabetes in Egypt is around 15.6% of all adults aged 20 to 79 (5). In 2013, Saudi Arabia was among the top ten countries of the world with highest prevalence where prevalence of diabetes was 23.9%
of total population (6).
Insulin is a heterodimeric polypeptide consisting of two chains, A and B, which have 21 and 30 amino acids, respectively. The two chains are linked together by two inter-chain disulfide bridges that connect A7 to B7 and A20 to B19. A third intra-chain disulfide bridge connects residues 6 and 11 of the A chain (7). Insulin is synthesized in β-cells of the pancreas as a preprohormone (molecular weight Received: 27 October 2016/ Revised: 20 February 2017 / Accepted: 10 March 2017 / Published: 02 March 2017
Abstract: Commercial therapeutic recombinant protein products have an important value as a treatment for human.
Human insulin is one of many examples of recombinant therapeutic proteins as it represents the essential treatment for all type І diabetes and a considerable number of type ІІ diabetic patients. Mini-proinsulin is known as a precursor of insulin that can be converted enzymatically into human insulin. In the current study, a synthetic gene coding for the insulin precursor was designed and synthesized. The synthetic gene was cloned into the cloning vector pCR4-TOPO. The gene was subcloned into pQE-30 Xa expression vector. The proinsulin-pQE-30 Xa was transformed into M15 bacteria followed by protein expression. Protein expression was evaluated using SDS-PAGE analysis. The identity of the expressed materials was evaluated using Western blot analysis. Results indicated that the expression system was good enough to express the pro-insulin and immunoblotting proved that the recovered protein is pro-insulin.
KKU Journal of Basic and Applied Sciences
approximately 11,500 Da) and is considered the prototype for peptides that are processed from larger precursor molecules.
Preproinsulin contains both an N-terminal signal peptide and an interstitial C-peptide (8). The function of the 23 hydrophobic amino acids in the leader sequence directs the molecule into the endoplasmic reticulum and then removed.
This action creates the proinsulin molecule (molecular weight 9 Da) that provides the conformation necessary for the proper disulfide bridges (Fig.1). After that, the molecule of proinsulin undergoes site-specific peptide cleavages at two specific trypsin sites which result in the formation of mature insulin and C-peptide (9).
Figure 1: The Structure of Human Proinsulin
Molecule. The Proteolytic cleavage sites are indicated by arrows (After Cowley & Mackin 1997).
C-peptide of the human is a 31-amino-acid that is a cleavage product of insulin biosynthesis. It was regarded as a biologically inert molecule that functions only to link and stabilize the A- and B-chains of insulin molecule, and as a result enables the correct folding and interchain disulfide bond formation (10).
There are two approaches to produce recombinant human insulin using prokaryotic system in E. coli as a host organism (11-13). In the first approach, which is referred as the two-chain or the non-enzymatic approach, is based on the separate production of insulin chains, A and B, fused to fusion protein partners to prevent any intracellular breakdown of the protein products. The A and B chains are then separately purified and subsequently combined in the form of stable S-sulfonate derivatives to generate native insulin (14-16). The other approach is the single-chain or enzymatic approach which is based on the bacterial synthesis of proinsulin which is fused to a fusion protein partner with either cyanogen bromide (12, 17, 18) or proteolytic cleavage site (19-22) to facilitate the separation of proinsulin from the fusion protein partner during further downstream processing.
The released proinsulin is subsequently refolded to form the correct disulfide linkages between the A and B chains and then the connecting C-peptide is enzymatically removed with the concomitant release of the native human insulin.
Proteolytic processing of proinsulin involved the combined utilization of trypsin and carboxypeptidase B(19-21). Further advances were introduced to increase the efficiency of enzymatic conversion of proinsulin to insulin leading to the improvement of the production yield of active human insulin from its precursor (23).
The 3-dimensional structure of insulin(24) shows that a peptide much shorter than the 35-amino acid connecting peptide of human proinsulin could be sufficient to connect the carboxy-terminus of the B chain with the amino-terminus of the A chain and thus might allow proper folding of such a modified proinsulin (25). In a study done by Chang et al (1998) of this mini-proinsulin, the 35-residue C-peptide was replaced by a short turn-forming peptide bridge composed of the nine amino acids (Arg-Arg-Tyr-Pro-Gly-Asp-Val-Lys- Arg), where the 2 basic moieties at the junctions (shown in bold) were conserved to facilitate the enzymatic conversion to active insulin (26). In addition, it was found that the process of refolding of the novel mini-proinsulin was much better by 20-40 % than that of normal proinsulin indicating that using of the short turn-forming sequence is more effective in the refolding process than using the longer C-peptide. It was successful to generate native human insulin by subsequent enzymatic conversion of mini-proinsulin (26).
In the current study, a bacterial expression system was established to produce human mini-proinsulin using an optimized synthetic gene.
2. MATERIALSANDMETHODS Gene design and synthesis
The gene coding for mini-proinsulin (PDB Accession#:
1EFE) was synthesized by Entelechon company (Gmbh, Germany) with adding the restriction sites BamHI and HindIII to the 5` and 3` ends respectively, cloned into pCR4-TOPO plasmid (Invitrogen) and designated as pCR4- TOPO/MPI. About 10 μg of the verified construct was lyophilized and supplied to the authors by Entelechon.
Sub-cloning of the mini-proinsulin gene
The lyophilized recombinant vector 1 (4 μg) was dissolved in 8 μl sterile TE buffer (pH 7.5, Sigma-Aldrich) to prepare a stock solution of 0.5 µg/μl final concentration. A 5 μl of the above stock solution was diluted ten times to prepare a working solution of 50 ng/μl final concentration. About 100 ng of plasmid DNA were used to transform competent DH5α E. coli cells (Promega).
After purification of the pCR4-TOPO/MPI recombinant plasmid by miniprep (Qiaprep spin miniprep kit, Qiagen), the gene of mini-proinsulin was released from pCR4- TOPO/MPI by digestion with the restriction endonucleases BamHI and HindIII enzymes. The released gene insert was purified from the reaction using the Qiaquick gel extraction kit (Qiagen) as described by the manufacturer. The purified insert was then ligated to BamHI and HindIII enzymes- digested and purified expression vector pQE-30 Xa and the resulting construct was given the name pMPI-T1 . The pMPI-T1 was used to transform competent JM109 cells which were prepared by the method described by Chung et al. (27). The presence of the pMPI-T1 in transformed bacteria was checked after plasmid extraction using the Qiaprep spin miniprep kit (Qiagene) and digestion with the endonucleases BamHI and HindIII using agarose gel electrophoresis. The results of restriction digestion were confirmed by PCR analysis. The construct pMPI-T1 was used to transform competent M15 bacteria. Successful transformants were checked for the presence of the insert using restriction endonuclease digestion and PCR analysis.
The PCR product of the expression construct was purified
KKU Journal of Basic and Applied Sciences
from the gel and the integrity of the gene insert was verified by DNA sequence analysis. Sequence alignment was carried out using the software Bio-Edit (version 7.0).
Expression of mini-proinsulin recombinant protein An overnight culture of the expression system M15[pMPI- T1] was prepared to be used in protein expression induction culture. A similar culture was prepared from the strain M15[pQE-40] to serve as a positive control for cell growth and expression conditions. A third culture was prepared from M15 cells transformed by the native pQE-30 Xa plasmid to serve as a negative control. Protein expression was induced in all cultures by adding IPTG to a final concentration of 1 mM. Samples of 1ml were collected from the expression cultures immediately before induction and at one hour intervals post-induction. Protein expression was analyzed using SDS-PAGE according to the method described by Laemmli (28) and following the protocols described by Sambrook & Russell (29).
Identity test for the expressed recombinant mini- proinsolin
Two healthy male New Zealand rabbits aged 3-6 months (body weight ~ 2.5 kg) lived in animal house at least for 3-4 days for adapting to the new circumstances were used for preparation of anti-human insulin antibodies according to Hu et al. (30) with some modifications. Briefly, 1 ml composed of 500 µl of human insulin solution and 500 µl of Complete Freund's Adjuvant emulsion was injected intradermally on the back and proximal limbs of the rabbits (30-50 μl per injection site). One week later, the immunisation was repeated three times with Incomplete Freund’s Adjuvant replacing Complete Freund’s Adjuvant at one week intervals. Five days after the last immunisation, blood was collected from the ear of the rabbit and titre of the anti-human insulin level was determined using radial immunnodiffusion method (31) using pre-immunization serum as negative control. The expressed proteins from the test culture (separated on the gel) were transferred onto a nitrocellulose membrane for 90 min at 50 Vas described by Towbin et al. (32). Immunodot blotting was carried out according to Kaufmann et al. (33). Nonspecific protein binding was reduced by preincubating the membrane overnight at 4°C inblocking buffer containing 5% BSA in TN buffer(10 mmol/l Tris, pH 7.2, and 0.9% NaCl). The membrane was then incubated with anti-human insulin antiserum diluted 1:200 in blocking bufferfor 1 h at 37°C.
The membrane was washed 5 times for 10 min in prewarmed TNTbuffer (TN buffer plus 0.1% Tween) with shaking. Goat anti-mouse alkaline phosphates conjugate (Sigma-Aldrich-USA) was dispensed to the membrane (dilute 1:3,000 in blocking buffer) and incubated with shaking for 1 hr at room temperature. After extensive washing, sufficient amount of NBT/BCIP solution (Roche) was added to cover the membrane. Once the immunoblots appeared on the nitrocellulose membrane, the reaction was stopped by adding distilled water. Water was then discarded, and the membrane was allowed for air dry.
3. RESULTS
Sub-cloning of the mini-proinsulin gene
The gene of mini-proinsulin released from the plasmid pCR4-TOPO/MPI was ligated to the vector pQE-30 Xa to form the expression construct pMPI-T1. After propagation in JM109 cells, the constructed plasmid was checked for successful gene cloning by restriction digestion analysis and PCR. Restriction digestion of the expression construct with BamHI and HindIII resulted in the release of the gene insert that showed a distinct band on agarose gel at 192 bp as expected (Fig. 2). The fragment of the construct comprising the insert was amplified by PCR using two primers specific for pQE vectors. Analysis of the PCR product showed a distinct band at the expected size of 367 bp (Fig. 3).
Preparation of the expression system and protein expression
M15 bacteria were transformed with the expression construct pMPI-T1. Analysis of the construct after the transformation was done by restriction digestion and PCR.
The integrity of the construct was tested by DNA sequence analysis of the fragment comprising the gene insert. Upon alignment, the resulting sequence was 100 % congruent with the expected gene sequence. Protein expression was induced by adding IPTG to the expression cultures. The expressed fusion protein showed a distinct band at approximately 10 kDa (Fig. 4). The positive control culture M15[pQE-40]
showed a distinct band of mouse DHFR at 26 kDa with intensity increasing with time (Fig. 5). The negative control culture M15[pQE-30 Xa] showed no bands in the range of 7-14 kDa (Fig. 6).
Figure 2: Restriction digestion analysis of the expression construct pMPI-T1 from JM109. Lane M: DNA molecular mass marker, Lane 1: BamHI & HindIII restriction products of the expression.
KKU Journal of Basic and Applied Sciences
Figure 3: PCR analysis of the expression construct pMPI-T1 from JM109. Lane M: DNA low molecular mass marker, Lane 1: negative control reaction, Lane 2: PCR product of the plasmid pQE-30 Xa (positive control) and Lane 3: PCR product of the expression construct.
Figure 4: SDS-PAGE of the expressed MPI-fusion protein in M15[pMPI-T1] expression system. Lane M1: Amersham low molecular weight standard, Lane C: un-induced control sample, lanes (1hr-6hr): samples taken at 1 hr time intervals after induction and Lane M2: RECOM pre-stained protein marker
Figure 5: SDS-PAGE of the expressed mouse DHFR protein (positive control). Lane M: broad range protein marker (NEB), Lane C: non-induced control sample, lanes 1he-5hr:
samples taken at 1 hr time intervals after induction.
Figure 6: SDS-PAGE of the total cell lysate of M15[pQE-30 Xa] E. coli (negative control). Lane C: un-induced control sample, lanes 1he-5hr: samples taken at 1 hr time intervals after induction. Lane M1: Amersham low molecular weight standard.
Identity test of the expressed protein:
Separated proteins were transferred to a nitrocellulose membrane and detected by immunoblotting technique using rabbit polyclonal antibodies against human insulin. The fusion protein showed positive blots with insulin antibodies at the expected molecular weight as shown in (Fig. 8).
Figure 7: Western blotting analysis of the recombinant fusion protein expressed in M15[pMPI-T1] expression system. Lane C: un-induced control sample, lanes 1he-6hr:
culture samples taken at 1 hr time intervals after induction.
The blots of the expressed fusion protein are indicated by the black arrow.
4. DISCUSSION
Preparation of expression systems for the production of commercially valuable proteins, like human insulin, was often not fully described in published reports, most probably to protect proprietary rights. In this study, the development of a prokaryotic expression system for the production of human mini-proinsulin was described in details. The synthesis of human insulin by recombinant DNA technology using E. coli can be accomplished by one of two approaches. The single-chain approach was selected in this study because only a single fermentation and a single isolation procedure are required to obtain proinsulin. In addition, proinsulin can be refolded more efficiently as compared to the two-chain approach(34). In order to increase the folding efficiency of the insulin precursor and the production yield of insulin, a mini-C analogue of human proinsulin was used in the current
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study as an insulin precursor instead of the normal proinsulin.
In this mini-proinsulin, the 31-residue central C-peptide region was replaced by a short peptide sequence. In previous studies, this was found to facilitate the refolding process and increase the refolding yield of this mini-proinsulin by 20-40
% better than that of proinsulin studied at the same molar concentration(26), showing that the short turn-forming sequence is more efficient in the process of refolding than the longer C-peptide. Native human insulin can be subsequently generated by enzymatic conversion of mini-proinsulin.
The gene required for the expression of heterologous proteins in E. coli can be either isolated from its native source or synthesized using DNA synthesis technology. In order to eliminate the need for tedious extraction of mRNA and construction of cDNA, DNA synthesis technology was utilized in the current study to synthesize the gene coding for mini-proinsulin. The use of synthetic genes is increasingly used now for the protein production because obtaining sequence information is easier than using the corresponding physical DNA (35). The increased speed and decreased cost of synthetic DNA provides a good way to obtain genes encoding heterologous proteins (36). In addition, the greatest advantage of the synthetic gene approach is the optimization of gene sequence for the expression in a specific target organism. Two different restriction sites were added to the 5' and 3' ends of the target gene to facilitate directional cloning of the gene into the expression vector. The two different restriction sites used were BamH I and Hind III. This is because the two sites were placed at the extreme ends of the multiple cloning site of the expression vector pQE-30 Xa, which will lower the amino acid number of the vector added to the expressed protein. BamHI and HindIII enzymes also work at 100 % efficiency in the same buffer which facilitates the restriction digestion of the gene insert by the two enzymes simultaneously in one reaction, thus, reducing the work labor.
The synthetic mini-proinsulin gene was subcloned into the expression vector pQE-30 Xa under the control of phage T5 promoter which is recognized by the RNA polymerase of E.
coli and the two lac operator sequences which increase the lac repressor binding and ensure successful repression of the powerful T5 promoter. Protein expression from the T5 promoter is induced by the synthetic lactose analogue IPTG instead of lactose. This is because IPTG is effective in small doses, the induction is not affected by the presence of glucose and the inducer is not metabolized by the cells(37). Some members of the pQE family of plasmids were previously employed for the expression of human proinsulin in E. coli.
For example, the vector pQE-31 was used for the expression of human proinsulin in XL2-blue(17) and M15 E. coli(18).
The pQE-30 Xa vector has the advantage of encoding a Factor Xa Protease recognition site which is bracketed by the 6xHis-tag coding region on the 5' side and the multiple-cloning site on the 3' side which facilitates the removal of the 6xHis-tag from the expressed recombinant protein. The mini-proinsulin gene was fused to the short fusion partner composed of six histidine moieties to facilitate further purification by immobilized metal affinity chromatography.
The expression construct was initially propagated in the strain JM109 because plasmid preparations derived from M15 will also contain the pREP4 plasmid, which could make clone analysis more difficult. In addition, the strain JM109 has a number of features that make it an ideal strain for the propagation and storage of pQE expression constructs.
Firstly, JM109 contains an episomal copy of lacIq gene, which is a mutation of lacI that produces very high levels of the lac repressor which efficiently blocks transcription of the cloned gene(38). JM109 also lacks the EcoK restriction system, so undesirable restriction of the cloned DNA is prevented. Moreover, during propagation in E. coli, DNA inserted into vectors is sometimes rearranged by the proteins involved in DNA recombination. Being a recombination -deficient strain (carrying the genotype recA-), JM109 is unable to recombine the cloned foreign gene to its chromosomal DNA(39).
After transformation of the expression construct pMPI-T1 into the strain M15, successful transformants were selected on LB agar containing ampicillin and kanamycin to ensure that both the expression construct and the pREP4 plasmid were retained in the grown transformants. Before passing to protein expression, the integrity of pMPI-T1 construct was tested by restriction digestion analysis and PCR analysis. The results were concordant with that obtained after propagation of the construct in the strain JM109. The correct orientation of the expression construct extracted from M15 was confirmed by DNA sequence analysis. DNA sequencing results showed that the MPI-T1 gene was successfully cloned into the expression vector in the correct orientation without any frame shifts. It was found that the whole sequence of the cloned gene was fully conserved and 100 % identical to the authentic gene sequence with no mutations.
The expressed fusion protein showed a distinct band at about 10 kDa as expected in the SDS-PAGE. The intensity of the band indicated that the fusion protein was produced at a relatively high yield compared to the total cell proteins indicating that this expression system is highly efficient, considering the relatively small size of the fusion protein.
The positive control expression culture M15[pQE-40]
showed a strong distinct band of mouse DHFR protein at 26 kDa with intensity increasing with time which indicated a good cell growth and expression conditions. In the same time, the negative control culture M15[pQE-30 Xa] showed no bands at 10 kDa confirming that the band appeared in the test expression culture was due to the gene product.
Immunoblotting analysis of expressed proteins showed that anti-human insulin antibodies could detect the expressed mini-proinsulin at its expected molecular weight. This indicated that the identity of the expressed protein was conserved during the cloning and sub-cloning process of the target gene.
5. CONCLUSION
In conclusion, the current study demonstrated a successful development of a bacterial expression system for the production of human mini-proinsulin using an optimized synthetic gene. The expression system used was efficient and can be considered as a promising starting point for the production of recombinant human insulin. With its relatively high expression yield, the novel mini-proinsulin used in this study as an insulin precursor can be an excellent alternative to the normal proinsulin, confirming the results previously obtained by other researchers.
KKU Journal of Basic and Applied Sciences ACKNOWLEDGMENT
Authors would like to thank The Holding Company for Biological Products and Vaccines (VACSERA), Cairo, Egypt, Research Center for Advanced Materials Science (RCAMS), King Khalid University, P.O. Box 9004, Abha 61413, Saudi Arabia, and National Organization for Research and Control of Biologicals, Cairo, Egypt for supporting this work.
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