# Patent

## Title
Method for enhancing extracellular secretion of recombinant proteins in Escherichia coli by co-expressing Thermobifida fusca cutinase

## Abstract

The present invention provides a method of increasing extracellular secretion of secretory proteins by co-expressing the secretory proteins with a mature cutinase. Cutinase can improve the permeability of E. coli cell membrane without destroying the membrane, and thus facilitate cross-membrane transfer of the secretory proteins co-expressed in E. coli. Increased extracellular secretion of target proteins can shorten cell culture time, reduce the formation of inclusion bodies and increase production of target proteins.

## Background
CROSS-REFERENCES AND RELATED APPLICATIONS
This application claims the benefit of priority of Chinese patent application serial no. 201210241426.7, entitled “A method for extracellular expression of Thermobifida fusca cutinase”, filed Jul. 5, 2012, and Chinese patent application serial no. 201210338830.6, entitled “A method for enhancing extracellular secretion of recombinant proteins and its application”, filed Sep. 3, 2012, which are herein incorporated by reference in entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for enhancing extracellular secretion of recombinant proteins, and more particularly relates to a method for enhancing extracellular secretion of recombinant proteins in Escherichia coli by co-expressing Thermobifida fusca (T. fusca) cutinase.
2. Description of the Related Art
Escherichia coli (E. coli) has a rapid growth rate and high protein expression level so that it has been considered as one of the most promising strain for industrial production of recombinant proteins. E. coli is able to express proteins in 3 ways: cytosolic production, periplasmic production and extracellular production. Compared with other two methods, extracellular production has significant advantages in both analytical and industrial applications because it does not require cell disruption and has almost no contamination from host proteins.
There are 5 secretory pathways for protein to translocate across the cell membrane in E. coli. However, due to their complicated components, regulatory mechanisms, and/or the low intrinsic secretion capabilities of these systems, E. coli is usually considered to be a poor secretor of proteins. It has been reported that the balance between expression level and secretion rate is a critical factor for extracellular production of recombinant proteins. To date, many strategies for improving the extracellular secretion of proteins in E. coli have been reported, especially the mechanism between transport chaperon protein and target proteins has been explored. Enhancing protein secretion process, constructing outer membrane leakage mutations as well as adding certain chemicals to enhance the permeability of cell membrane are commonly used methods.
Cutinase not only catalyzes the cleavage of the ester bonds of cutins, but also is capable of hydrolyzing soluble esters, insoluble triglycerides and a variety of polyesters. In addition to its hydrolytic activity, cutinase is also used in ester synthesis and transesterification. As a multifunctional enzyme, cutinase has potential in the food, chemical, textile, and other industries. Enzymes with cutinase activity have been found in both fungi and bacteria. To date, all the cutinase from microorganisms have been found to be secretory enzymes. Therefore, for the heterologous expression, signal peptides are usually used to mediate the secretion of recombinant cutinase. Using this approach, Fusarium solani cutinase has been expressed in a variety of host cells. The highest yield of extracellular protein 546 mg/L, was obtained in a 5 L bioreactor using an engineered Saccharomyces cerevisiae cellular system. Previously, we identified the open reading frame responsible for the expression of T. fusca cutinase, and secretory expression of the cutinase was performed by mediation of the pelB signal peptide in E. coli BL21 (DE3). More recently, we found that the mature form of T. fusca cutinase without a signal peptide was mainly secreted into the culture medium and only a small percentage of the enzyme is located in the periplasm and cytoplasm. We further found that the mature cutinase has phospholipase activity that leads to limited hydrolysis of phospholipids of cell membrane and thus increases membrane permeability. Based on this finding, the present invention provides a method for extracellular secretion of mature cutinase without mediation of a signal peptide and a method for enhancing extracellular secretion of recombinant proteins. Mature cutinase used herein refers to a full-length cutinase protein without a signal peptide.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Time profiles for the production of cutinase by E. coli BL21 (DE3)/Tfu—0883/pET-20b (+)NS in a shaking flask. ▪, Dry cell weight (DCW); □, cutinase activity in the culture supernatant.
FIG. 2. SDS-PAGE analysis of the expression of cutinase by E. coli BL21 (DE3)/Tfu—0883/pET-20b (+)NS. M, molecular mass standard protein; 1, culture supernatant; 2, intracellular soluble fraction; 3, intracellular insoluble fraction.
FIG. 3. Time profiles for the production of cutinase by E. coli BL21 (DE3)/Tfu—0883/pET-20 b (+)NS in a 3 L fermentor. ▪, DCW; □, cutinase activity in the culture supernatant.
FIG. 4. Time profiles for the production of α-amylase by E. coli BL21 (DE3). ▪, the recombinant E. coli BL21 (DE3) harboring amy/pET-20b(+) as control, ● E. coli BL21 (DE3) harboring Tfu—0883/amy/pETDuet-1.
FIG. 5. SDS-PAGE analysis of the expression of α-amylase. M, molecular mass standard protein; 1, culture supernatant of E. coli BL21 (DE3) harboring amy/pET-20b(+); 2, culture supernatant of E. coli BL21 (DE3) harboring Tfu—0883/amy/pETDuet-1.
FIG. 6. Time profiles for the production of xylanase by E. coli BL21 (DE3). ▪, E. coli BL21 (DE3) harboring xynA/pET-20b(+); ●, E. coli BL21 (DE3) harboring Tfu—0883/xynA/pETDuet-1.
FIG. 7. SDS-PAGE analysis of the expression of xylanase. M, molecular mass standard protein; 1, extracellular fraction of E. coli BL21 (DE3) harboring xynA/pET-20b(+); 2, extracellular fraction of E. coli BL21 (DE3) harboring Tfu—0883/xynA/pETDuet-1; 3, intracellular soluble fraction of E. coli BL21 (DE3) harboring xynA/pET-20b(+); 4, intracellular soluble fraction of E. coli BL21 (DE3) harboring Tfu—0883/xynA/pETDuet-1; 5, intracellular insoluble fraction of E. coli BL21 (DE3) harboring xynA/pET-20b (+); 6, intracellular insoluble fraction of E. coli BL21 (DE3) harboring Tfu—0883/xynA/pETDuet-1.



DETAILED DESCRIPTION
The present invention provides a method for extracellular secretory expression of Thermobifida fusca cutinase (T. fusca cutinase, NCBI accession number AAZ54921) in E. coli without mediation of a signal peptide and a method for enhancing extracellular secretion of recombinant proteins in E. coli by co-expressing mature T. fusca cutinase. The methods mainly comprise plasmid construction without any signal peptide, expressing T. fusca cutinase and co-expressing target proteins with T. fusca cutinase.
The method for secretory expression of T. fusca cutinase in E. coli without mediation of a signal peptide comprises the following steps:
1. Plasmid Construction
The mature cutinase gene without the signal peptide sequence (124-906 bp in the gene sequence of NCBI accession number AAZ54921) was amplified from Tfu—0883/pET-20b(+) (Sheng Chen, Xing Tong, Ronald W. Woodard, Guocheng Du, Jing Wu and Jian Chen. Identification and Characterization of Bacterial Cutinase. Journal of Biological Chemistry. 2008. 283(38): 25854-62.) by a polymerase chain reaction. The PCR product was isolated, amplified and ligated into the expression vector pET-20b(+). The resulting recombinant plasmid Tfu—0883/pET-20b(+)NS, which has a full-length T. fusca cutinase gene without any signal peptide, was verified by DNA sequencing.
2. Expression of Recombinant Cutinase
The recombinant plasmid Tfu—0883/pET-20b(+)NS was transformed into E. coli, and the transformed E. coli cells were cultivated and induced to express cutinase. The culture broth was centrifuged and the supernatant was collected for purification of cutinase.
The E. coli host strain could be any of E. coli BL21 (DE3), E. coli W3110, E. coli JM109, E. coli JM109 (DE3) and E. coli DH5α, among which the E. coli BL21 (DE3) was preferred.
The vector used for protein expression could be any of pUC, pET, pT7-7, and pGEX.
The method for enhancing extracellular secretion of recombinant proteins by co-expressing T. fusca cutinase comprises the following steps:
1. Plasmid Construction
The Tfu—0883/pETDuet-1 and the recombinant plasmid pET-20b (+) containing a gene encoding a target protein were excised using two restriction enzymes. And the target protein gene was ligated into Tfu—0883/pETDuet-1 to make a Tfu—0883/target protein/pETDuet-1 plasmid. The Tfu—0883/target protein/pETDuet-1 plasmid, which contains genes for mature T. fusca cutinase and the target protein, was amplified in E. coli JM109.
2. Expression of Target Protein
The Tfu—0883/target protein/pETDuet-1 plasmid containing genes encoding T. fusca cutinase and the target protein was transformed into E. coli and the transformed E. coli was cultivated for target protein expression. After that, the culture broth was centrifuged and the supernatant was collected for target protein purification.
The host strain for expression could be any of E. coli BL21 (DE3), E. coli W3110, E. coli JM109, E. coli JM109 (DE3) and E. coli DH5α, among which the E. coli BL21 (DE3) was preferred.
The vector used for expression could be any of pETDuet-1, pCOLADuet-1, pUC, pET, pT7-7 and pEGX.
The target proteins are secretory proteins that are synthesized in the cytoplasm and, with the help of transport proteins, are transferred across inner and outer cell membrane, and secreted into the culture medium through a secretion pathway. The secretion pathway can be Type I, II, III, IV, and V secretory pathway in E. coli cells. The Type II secretion pathway mediated by SecB is preferred. The target proteins can be, for example, intrinsic E. coli secretory proteins or proteins with an E. coli signal peptide for extracellular secretion.
The method of constructing Tfu—0883/pETDuet-1 that contains cutinase gene from T. fusca was described in Chinese patent application no. 201210045965.3, which is herein incorporated by reference in its entirety.
The present invention uses mature cutinase to significantly improve the permeability of E. coli cell membrane and enhance the extracellular secretion of recombinant proteins without destroying the cell membrane, which can significantly decrease formation of inclusion bodies, shorten cell culture time, and increase productivity. This method has important applications in large scale production of industrial proteins.
EXAMPLES
The following examples were provided by way of illustration only, and not by way of limitation. Standard experimental operations not specifically described in the specification were preformed according to standard molecular cloning protocols described in “Molecular Cloning: A Laboratory Manual” (by Sambrook J and Russell D, Cold Spring Harbor Laboratory Press, 2001) .
Strains, Vectors and Materials:
Streptomyces sp. FA1 and Bacillus licheniformis were from China Center for Type Culture Collection (CCTCC), and the CCTCC accession numbers were M 2012310 and M 2013012, respectively. The strain T. fusca (ATCC 27730) was obtained from ATCC. E. coli JM109 strain was used as the host for gene cloning and E. coli BL21 (DE3) strain was used for protein expression.
The plasmid Tfu—0883/pET-20b (+) (Sheng Chen, Xing Tong, Ronald W. Woodard, Guocheng Du, Jing Wu and Jian Chen. Identification and Characterization of Bacterial Cutinase. Journal of Biological Chemistry. 2008. 283(38): 25854-62.) harboring the gene of T. fusca cutinase (NCBI accession number AAZ54921) and purified wild-type cutinase from the culture medium of T. fusca were laboratory stock. The pET-20 b (+) and pETDuet-1 vector from Novagen (a company of EMD Chemicals, Philadelphia, Pa., USA) were utilized as expression vectors.
The restriction enzymes, alkaline phosphatase, T4 DNA ligase, agarose gel DNA purification kit and pMD18-T simple vector were obtained from TakaRa (Dalian, China). The DNA sequencing and primer synthesis were performed by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd (Shanghai, China). The 4-Nitrophenyl butyrate (pNPB) was obtained from Sigma (Shanghai, China). Other chemicals were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).
Example 1
Construction of Tfu—0883/pET-20b(+)NS Plasmid
Construction of Tfu—0883/pET-20b(+)NS plasmid comprised the following steps:
(1) The gene encoding a mature cutinase (124-906 bp in the gene sequence of NCBI accession number AAZ54921) was amplified from Tfu—0883/pET-20b (+), which contained the full length cutinase gene of NCBI accession number AAZ54921 inserted in Nco I and EcoR I restriction enzyme sites of plasmid pET-20b), by polymerase chain reaction using the primers P1 (SEQ ID No: 1) and P2 (SEQ ID No: 2) in 50 μL PCR system. SEQ ID No. 1: gTAATCCATATggCCAACCCCTACgAgCgC (With restriction enzyme Nde I cutting site). SEQ ID No. 2: AgAgAATTCgggAACgggCAggTggAgCg (With restriction enzyme EcoR I cutting site). The PCR process was as follows: denaturation at 94° C. for 4 min, cyclical reaction for 30 times (98° C. for 10 s, 60° C. for 5 s, 72° C. for 1 min), elongation at 72° C. for 10 min, and finally being kept at 4° C. After gel purification, the PCR products were ligated into pMD18-T Simple vector and then transformed into E. coli JM109. The transformation products were spread on LB agar plate containing ampicllin (LB-Amp) for overnight cultivation at 37° C. The single colony was transferred into liquid LB for plasmid amplification. The recombinant plasmid was then verified by DNA sequencing.
(2) A plasmid with the correct sequence of the T. fusca cutinase gene and vector pET-20b(+) were separately digested with the restriction enzymes, Nde I (Nde I has a restriction enzyme cutting site located upstream of intrinsic pelB signal peptide) and EcoR I. To get the recombinant plasmid Tfu—0883/pET-20b (+)NS, the gel band purification products were then ligated by T4 DNA ligase at 16° C. for overnight. And then, the recombinant plasmids were transformed into E. coli JM109. The transformation products were spread on LB-Amp agar plate for overnight cultivation at 37° C. The single colony was transferred into liquid LB-Amp for plasmid amplification. 8-10 h later, plasmids Tfu—0883/pET-20b (+)NS were extracted, purified and verified by DNA sequencing.
Example 2
Expression of Tfu—0883/pET-20b(+)NS by E. coli BL21 (DE3)
Expression of Tfu—0883/pET-20b(+)NS by E. coli BL21 (DE3) comprised the following steps:
After transformation, E. coli BL21 (DE3) containing Tfu—0883/pET-20b (+)NS was cultivated on LB-Amp agar plate for overnight at 37° C. The single colonies were transferred into liquid LB-Amp and cultivated to later logarithmic phase. The culture broth was inoculated into liquid TB (Containing 100 μg/mL Amp) with 5% inoculation percent and grown at 37° C. for 2 h. Lactose was added to a final concentration of 5 g/L and the temperature was lowered to 25° C. for induction.
As shown in FIG. 1, during cultivation in the shake flask, the cutinase activity in the culture medium increased rapidly from 12 h of post induction and reached a maximum of 204.8 U/mL at 24 h. However, the cutinase activity in the cytoplasm was only 10.1 U/mL at the same time, which meant the cutinase without a signal peptide was almost completely “secreted” into the medium. The distribution of the cutinase was investigated by SDS-PAGE. As shown in FIG. 2, one major band around 31 kDa which is in agreement with the calculated molecular weight of mature cutinase, was found in the culture medium. In addition, no visible band was observed in either the cytoplasm or the periplasm.
Example 3
3 L Bioreactor for Production of Cutinase
The extracellular “secretion” efficiency of the above E. coli BL21 (DE3) harboring cutinase was further explored in 3 L fermentor. Exponential fed-batch culture strategy at a controlled speed was carried out to produce cutinase using recombinant E. coli BL21 (DE3) high cell-density culture.
The 3 L bioreactor for production of cutinase comprised the following steps:
(1) During fermentation, when the initial carbon resource (Glycerol) worked out at 6 h, the fresh substrate was fed. The feed was exponentially increased and coincided with specific growth rate (μ=0.25 h−1).
(2) When the OD600 of culture broth reached 50, lactose was added for induction at a flow rate of 0.6 g/L/hr.
As shown in FIG. 3, the cutinase activity in the culture medium increased rapidly from 9 h of post induction and reached a maximum of 1063 U/mL that was 5.2 times higher than in the shake flask cultivation. In addition, the productivity 44.2 U/mL/h obtained by this way was 1.9 times than that obtained by utilizing secretory pathway Type I and represented the highest productivity reported to date. The excellent yield achieved in 3 L bioreactor by expressing T. fusca cutinase in E. coli without mediation of a signal peptide makes it possible that cutinase can be produced in industrial scale.
Example 4
Co-Express α-Amylase with T. fusca Cutinase
The method of co-expressing α-amylase with T. fusca cutinase comprises the following steps:
(1) Construction of recombinant plasmid Tfu—0883/amy/pETDuet-1. Plasmid Tfu—0883/pETDuet-1 containing gene encoding mature T. fusca cutinase inserted in Nco I and Hind III restriction enzyme sites of the first multiple cloning site of pETDuet-1, and plasmid amy/pET-20b (+) (Described in detail in Chinese patent application serial number: 201210274173.3) containing gene encoding α-amylase inserted in Nco I and Hind III restriction enzyme sites of pET-20b (+) were separately digested with the restriction enzymes, Nde I and Xho I. The gel band purification products were then ligated by T4 DNA ligase at 16° C. for overnight. And then, the recombinant plasmids were transformed into E. coli JM109. The transformation products were spread on LB (Containing 100 mg/L amp) agar plate for overnight cultivation at 37° C. The single colonies were transferred into liquid LB-Amp for plasmid amplification. The recombinant plasmid Tfu—0883/amy/pETDuet-1 containing genes of T. fusca cutinase and a-amylase inserted in the first and second multiple cloning site of pETDuet-1, respectively, was extracted, purified and verified by DNA sequencing.
(2) Expression of α-amylase. After transformation, E. coli BL21 (DE3) containing Tfu—0883/amy/pETDuet-1 were cultivated on LB-Amp agar plate at 37° C. for 8-10 hr. The single colonies were transferred into liquid LB-Amp and cultivated at 37° C. for 8-10 hr. The culture broth was inoculated into liquid TB (Containing 5 g/L glycerol, 12 g/L peptone, 24 g/L yeast extract, 12.54 g/L K2HPO4, 2.31 g/L KH2PO4 and 100 μg/mL Amp) with 5% inoculation percent and grown at 37° C. for 2 hr. Then, IPTG was added to the culture broth with a final concentration of 0.4 mM. The culture was continued for another 60 hr after induction at 30° C. The recombinant E. coli BL21 (DE3) harboring amy/pET-20b (+) expressing the individual target enzyme was cultured under the same conditions as control.
(3) Enzymatic activity assay. The supernatant which was derived from separating cells by centrifugation at 8000 rpm for 10 min at 4° C. was used to measure the α-amylase activity in the culture medium. As shown in FIG. 4, the maximum α-amylase activity in the culture medium was 308 U/mL when α-amylase was expressed alone, and 358 U/mL when α-amylase was co-expressed with cutinase. The yield of co-expression system was higher by 16.2%, but because of the shortened cultivation period by half, the productivity was improved significantly. In addition, no α-amylase activity was detected in the intracellular fragments at the end of the cultivation in either case, suggesting that α-amylase was totally secreted into the culture medium. SDS-PAGE analysis (FIG. 5) of the culture medium showed that one distinct band for α-amylase of similar molecular weight, about 53 kDa, was found in the culture medium in both cases.
Example 5
Co-Express Zylanase with T. fusca Cutinase
The method of co-expressing xylanase with T. fusca cutinase comprised the following steps:
(1) Construction of recombinant plasmid Tfu—0883/xynA/pETDuet-1. Plasmid Tfu—0883/pETDuet-1 containing gene encoding T. fusca cutinase and plasmid xynA/pET-20 b (+) (Described in detail in Chinese patent document, application number: 201210284346.X) containing gene encoding xylanase inserted in Nco I and EcoR I restriction enzyme sites of pET-20 b (+) were separately digested with the restriction enzymes, Nde I and Xho I. The gel band purification products were then ligated by T4 DNA ligase at 16° C. for overnight. And then, the recombinant plasmids were transformed into E. coli JM109. The transformation products were spread on LB (Containing 100 mg/L amp) agar plate for overnight cultivation at 37° C. The single colonies were transferred into liquid LB-Amp for plasmid amplification. plasmids The recombinant plasmid Tfu—0883/xynA/pETDuet-1 containing genes of T. fusca cutinase and xylanase inserted in the first and second multiple cloning site of pETDuet-1, respectively, were extracted, purified and verified by DNA sequencing.
(2) Expression of xylanase. After transformation, E. coli BL21 (DE3) containing Tfu—0883/xynA/pETDuet-1 was cultivated on LB-Amp (Containing 100 mg/L amp) agar plate at 37° C. for 8-10 h. The single colonies were transferred into liquid LB-Amp and cultivated at 37° C. for 8-10 h. The culture broth was inoculated into liquid TB (Containing 5 g/L glycerol, 12 g/L peptone, 24 g/L yeast extract, 12.54 g/L K2HPO4, 2.31 g/L KH2PO4 and 100 μg/mL Amp) with 5% inoculation percent and grown at 25° C. for 6 h. Then, IPTG was added to the culture broth with a final concentration of 0.1 mM. The culture was continued for another 60 h after induction. The recombinant E. coli BL21 (DE3) harboring xynA/pET-20b (+) expressing the individual target enzyme was cultured under the same conditions as control.
(3) Enzymatic activity assay. The supernatant which was derived from separating cells by centrifugation at 8000 rpm for 10 min at 4° C. was used to measure the xylanase activity in the culture medium. As shown in FIG. 6, the maximum xylanase activity in the culture medium was 68 U/mL when xylanase was expressed alone, and 480 U/mL when xylanase was co-expressed with cutinase. The yield of co-expression system was higher by 6-fold, and the cultivation period was shortened by 10 h. In addition, at the end of the cultivation, the intracellular xylanase activity was 5.2 U/mL for the cells expressing xylanase alone and 8.8 U/mL for the co-expression system, suggesting that the majority of soluble xylanase had been secretes into the culture medium in both cases. Notably, SDS-PAGE analysis (FIG. 7) demonstrated the presence of inclusion bodies in the E. coli cells expressing xylanase alone, unlike that of the E. coli used in the co-expression system. The results presented here demonstrated that co-expressing cutinase accelerated the transport rate of target proteins, thus, the balance between expression level and secretion rate was achieved. Correspondingly, fewer inclusion bodies were observed by SDS-PAGE.
While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. All figures, tables, appendices, patents, patent applications and publications, referred to above, are hereby incorporated by reference.

## Claims

1. A method for secretory expression of T. fusca cutinase in E. coli without mediation of a signal peptide, comprising cloning a mature cutinase gene from T. fusca into an expression vector that does not comprise a signal peptide, expressing said mature cutinase in an E. coli host strain and obtaining said T. fusca cutinase from the culture supernatant.

2. The method of claim 1, comprising the steps of:
1) Amplify a gene encoding mature T. fusca cutinase from plasmid Tfu—0883/pET-20b(+) and ligate said T. fusca cutinase gene into vector pET-20b(+) to make a plasmid Tfu—0883/pET-20b(+)NS, wherein the pelB signal peptide of pET-20b(+) plasmid is removed from said plasmid Tfu—0883/pET-20b(±)NS;
2) Transform said plasmid Tfu—0883/pET-20b (+)NS into an E. coli host strain; and
3) Culture said E. coli harboring Tfu—0883/pET-20b(+)NS, induce secretory expression of T. fusca cutinase, and obtain T. fusca cutinase from the culture supernatant.

3. The method of claim 1, wherein said E. coli host strain is selected from the group consisting of E. coli BL21 (DE3), E. coli W3110, E. coli JM109, E. coli JM109 (DE3) and E. coli DH5α.

4. The method of claim 2, wherein said E. coli host strain is selected from the group consisting of E. coli BL21 (DE3), E. coli W3110, E. coli JM109, E. coli JM109 (DE3) and E. coli DH5α.

5. The method of claim 1, wherein said host strain is E. coli BL21 (DE3).