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A novel methodology for enhanced and consistent heterologous expression of unmodified human cytochrome P450 1B1 (CYP1B1).

Faiq MA, Ali M, Dada T, Dada R, Saluja D - PLoS ONE (2014)

Bottom Line: However, the drawback of these studies is that it changes the original protein and, as a result, invalidates functional studies.We aimed at expressing CYP1B1 in various strains of E. coli and in the course developed a protocol that results in high expression of unmodified protein sufficient for functional/biophysical studies.Though employed for CYP1B1 expression, this protocol can ideally be used to express any eukaryotic membrane protein.

View Article: PubMed Central - PubMed

Affiliation: Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India; Medical Biotechnology Laboratory, Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi, North Campus, Delhi, India; Laboratory for Molecular Reproduction and Genetics, Department of Anatomy, All India Institute of Medical Sciences, Ansari Nagar, India.

ABSTRACT
Cytochrome P450 1B1 (CYP1B1) is a universal cancer marker and is implicated in many other disorders. Mutations in CYP1B1 are also associated with childhood blindness due to primary congenital glaucoma (PCG). To understand the CYP1B1 mediated etiopathology of PCG and pathomechanism of various cancers, it is important to carry out its functional studies. Heterologous expression of CYP1B1 in prokaryotes is imperative because bacteria yield a higher amount of heterologous proteins in lesser time and so the expressed protein is ideal for functional studies. In such expression system there is no interference by other eukaryotic proteins. But the story is not that simple as expression of heterologous CYP1B1 poses many technical difficulties. Investigators have employed various modifications/deletions of CYP N-terminus to improve CYP1B1 expression. However, the drawback of these studies is that it changes the original protein and, as a result, invalidates functional studies. The present study examines the role of various conditions and reagents in successful and consistent expression of sufficient quantities of unmodified/native human CYP1B1 in E. coli. We aimed at expressing CYP1B1 in various strains of E. coli and in the course developed a protocol that results in high expression of unmodified protein sufficient for functional/biophysical studies. We examined CYP1B1 expression with respect to different expression vectors, bacterial strains, types of culture media, time, Isopropyl β-D-1-thiogalactopyranoside concentrations, temperatures, rotations per minute, conditioning reagents and the efficacy of a newly described technique called double colony selection. We report a protocol that is simple, easy and can be carried out in any laboratory without the requirement of a fermentor. Though employed for CYP1B1 expression, this protocol can ideally be used to express any eukaryotic membrane protein.

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Schematic Diagram of CYP1B1 gene.The CYP1B1 gene has 2 introns and 3 exons. The open reading frame starts from Exon II and ends within Exon III. The gene codes for a 543 aminoacid protein with a membrane spanning domain at the N-terminal followed by a proline rich hinge region which in turn is followed by the cytosolic globular domain. The chromosomal location of the gene is 2p21–22.
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pone-0110473-g001: Schematic Diagram of CYP1B1 gene.The CYP1B1 gene has 2 introns and 3 exons. The open reading frame starts from Exon II and ends within Exon III. The gene codes for a 543 aminoacid protein with a membrane spanning domain at the N-terminal followed by a proline rich hinge region which in turn is followed by the cytosolic globular domain. The chromosomal location of the gene is 2p21–22.

Mentions: Eukaryotic membrane proteins are difficult to express in heterologous hosts. This is especially true for transmembrane proteins [34]. CYP1B1 expression is particularly difficult in this regard and, therefore, several studies have been carried out using N-terminal modified protein [22], [23], [25]–[29]. Hence, it is necessary to develop a protocol that expresses the full length protein in sufficient quantities for functional studies. Additionally, proline is thought to be poorly translated in E. coli independent of its position in the translation product and regardless of the codon bias [35]. Interestingly, CYP1B1 has a proline rich hinge region towards the N-terminal region near the transmembrane domain to allow flexibility (Fig. 1). This phenomenon presumably further contributes towards the difficulties encountered in heterologous CYP1B1 expression. Investigators reporting CYP1B1 expression have circumvented these hindrances by N-terminal modifications [22], [23]. This is because N-terminal modifications have been reported to enhance heterologous expression of eukaryotic membrane proteins. A reasonable explanation for this observation is that eukaryotic mechanisms for translation initiation are extensively dissimilar to that of prokaryotes [36]. Hence, N-terminal sequences seem to be an important decisive factor in heterologous protein expression and that is why investigators pay attention to its modification. Literature suggests that initial 15–25 codons of the open reading frame deserve extensive deliberation for optimization of expression [37]–[45]. Such a method yields good expression but N-terminal specific functions of the protein are lost. As a representative instance, it will be conceptually warranted to mention here that the founder mutation G61E (an N-terminal mutation contiguous to the N-terminal proline rich hinge area) is deleterious to protein function and results in disease phenotype [46]. It is the proline-proline-glycine-proline motif that is presumably involved in joining of the N-terminal region of CYP1B1 with the globular domain for proper functioning of the CYP1B1 protein [17], [47]–[49]. Such effects may change a number of characteristics of the expressed protein involving cell signaling, enzymatic activities, functional characteristics, protein stability, charge, structure and many other functions. It is, therefore, always desirable to express the protein of interest with its native aminoacid sequence. This becomes particularly important in mutational studies and more so when the mutations are in the N-terminal region. Inspired by this reasoning, we undertook the important task of expressing native (unmodified) CYP1B1 in sufficient quantities in E. coli.


A novel methodology for enhanced and consistent heterologous expression of unmodified human cytochrome P450 1B1 (CYP1B1).

Faiq MA, Ali M, Dada T, Dada R, Saluja D - PLoS ONE (2014)

Schematic Diagram of CYP1B1 gene.The CYP1B1 gene has 2 introns and 3 exons. The open reading frame starts from Exon II and ends within Exon III. The gene codes for a 543 aminoacid protein with a membrane spanning domain at the N-terminal followed by a proline rich hinge region which in turn is followed by the cytosolic globular domain. The chromosomal location of the gene is 2p21–22.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199734&req=5

pone-0110473-g001: Schematic Diagram of CYP1B1 gene.The CYP1B1 gene has 2 introns and 3 exons. The open reading frame starts from Exon II and ends within Exon III. The gene codes for a 543 aminoacid protein with a membrane spanning domain at the N-terminal followed by a proline rich hinge region which in turn is followed by the cytosolic globular domain. The chromosomal location of the gene is 2p21–22.
Mentions: Eukaryotic membrane proteins are difficult to express in heterologous hosts. This is especially true for transmembrane proteins [34]. CYP1B1 expression is particularly difficult in this regard and, therefore, several studies have been carried out using N-terminal modified protein [22], [23], [25]–[29]. Hence, it is necessary to develop a protocol that expresses the full length protein in sufficient quantities for functional studies. Additionally, proline is thought to be poorly translated in E. coli independent of its position in the translation product and regardless of the codon bias [35]. Interestingly, CYP1B1 has a proline rich hinge region towards the N-terminal region near the transmembrane domain to allow flexibility (Fig. 1). This phenomenon presumably further contributes towards the difficulties encountered in heterologous CYP1B1 expression. Investigators reporting CYP1B1 expression have circumvented these hindrances by N-terminal modifications [22], [23]. This is because N-terminal modifications have been reported to enhance heterologous expression of eukaryotic membrane proteins. A reasonable explanation for this observation is that eukaryotic mechanisms for translation initiation are extensively dissimilar to that of prokaryotes [36]. Hence, N-terminal sequences seem to be an important decisive factor in heterologous protein expression and that is why investigators pay attention to its modification. Literature suggests that initial 15–25 codons of the open reading frame deserve extensive deliberation for optimization of expression [37]–[45]. Such a method yields good expression but N-terminal specific functions of the protein are lost. As a representative instance, it will be conceptually warranted to mention here that the founder mutation G61E (an N-terminal mutation contiguous to the N-terminal proline rich hinge area) is deleterious to protein function and results in disease phenotype [46]. It is the proline-proline-glycine-proline motif that is presumably involved in joining of the N-terminal region of CYP1B1 with the globular domain for proper functioning of the CYP1B1 protein [17], [47]–[49]. Such effects may change a number of characteristics of the expressed protein involving cell signaling, enzymatic activities, functional characteristics, protein stability, charge, structure and many other functions. It is, therefore, always desirable to express the protein of interest with its native aminoacid sequence. This becomes particularly important in mutational studies and more so when the mutations are in the N-terminal region. Inspired by this reasoning, we undertook the important task of expressing native (unmodified) CYP1B1 in sufficient quantities in E. coli.

Bottom Line: However, the drawback of these studies is that it changes the original protein and, as a result, invalidates functional studies.We aimed at expressing CYP1B1 in various strains of E. coli and in the course developed a protocol that results in high expression of unmodified protein sufficient for functional/biophysical studies.Though employed for CYP1B1 expression, this protocol can ideally be used to express any eukaryotic membrane protein.

View Article: PubMed Central - PubMed

Affiliation: Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India; Medical Biotechnology Laboratory, Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi, North Campus, Delhi, India; Laboratory for Molecular Reproduction and Genetics, Department of Anatomy, All India Institute of Medical Sciences, Ansari Nagar, India.

ABSTRACT
Cytochrome P450 1B1 (CYP1B1) is a universal cancer marker and is implicated in many other disorders. Mutations in CYP1B1 are also associated with childhood blindness due to primary congenital glaucoma (PCG). To understand the CYP1B1 mediated etiopathology of PCG and pathomechanism of various cancers, it is important to carry out its functional studies. Heterologous expression of CYP1B1 in prokaryotes is imperative because bacteria yield a higher amount of heterologous proteins in lesser time and so the expressed protein is ideal for functional studies. In such expression system there is no interference by other eukaryotic proteins. But the story is not that simple as expression of heterologous CYP1B1 poses many technical difficulties. Investigators have employed various modifications/deletions of CYP N-terminus to improve CYP1B1 expression. However, the drawback of these studies is that it changes the original protein and, as a result, invalidates functional studies. The present study examines the role of various conditions and reagents in successful and consistent expression of sufficient quantities of unmodified/native human CYP1B1 in E. coli. We aimed at expressing CYP1B1 in various strains of E. coli and in the course developed a protocol that results in high expression of unmodified protein sufficient for functional/biophysical studies. We examined CYP1B1 expression with respect to different expression vectors, bacterial strains, types of culture media, time, Isopropyl β-D-1-thiogalactopyranoside concentrations, temperatures, rotations per minute, conditioning reagents and the efficacy of a newly described technique called double colony selection. We report a protocol that is simple, easy and can be carried out in any laboratory without the requirement of a fermentor. Though employed for CYP1B1 expression, this protocol can ideally be used to express any eukaryotic membrane protein.

Show MeSH
Related in: MedlinePlus