CODON OPTIMIZATION INCREASES HUMAN

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Dec 9, 2012 - The output of opti-kallistatin protein was $2-fold increase (2.09 Æ ... mon codon optimization in the E. coli host significantly increases the yield ...
This article was downloaded by: [Sun Yat-Sen University], [Guoquan Gao] On: 09 December 2012, At: 23:09 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

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CODON OPTIMIZATION INCREASES HUMAN KALLISTATIN EXPRESSION IN Escherichia coli a

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a

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Zhiyu Dai , Yifei Chen , Weiwei Qi , Lijun Huang , Yang Zhang a

, Ti Zhou , Xia Yang

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& Guoquan Gao

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Department of Biochemistry, Zhongshan Medical School, Sun Yatsen University, Guangzhou, Guangdong Province, China b

Department of Medical Laboratory, Guangdong General Hospital, Guangzhou, Guangdong Province, China c

China Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Ministry of Education, Guangzhou, Guangdong Province, China d

Department of Education of Guangdong Province, Key Laboratory of Functional Molecules from Marine Microorganisms (Sun Yat-sen University), Guangzhou, Guangdong Province, China Version of record first published: 07 Dec 2012.

To cite this article: Zhiyu Dai , Yifei Chen , Weiwei Qi , Lijun Huang , Yang Zhang , Ti Zhou , Xia Yang & Guoquan Gao (2013): CODON OPTIMIZATION INCREASES HUMAN KALLISTATIN EXPRESSION IN Escherichia coli , Preparative Biochemistry and Biotechnology, 43:1, 123-136 To link to this article: http://dx.doi.org/10.1080/10826068.2012.712079

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Preparative Biochemistry & Biotechnology, 43:123–136, 2013 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2012.712079

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CODON OPTIMIZATION INCREASES HUMAN KALLISTATIN EXPRESSION IN Escherichia coli

Zhiyu Dai,1 Yifei Chen,1 Weiwei Qi,1 Lijun Huang,1 Yang Zhang,2 Ti Zhou,1 Xia Yang,1,4 and Guoquan Gao1,3 1 Department of Biochemistry, Zhongshan Medical School, Sun Yat-sen University, Guangzhou, Guangdong Province, China 2 Department of Medical Laboratory, Guangdong General Hospital, Guangzhou, Guangdong Province, China 3 China Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Ministry of Education, Guangzhou, Guangdong Province, China 4 Department of Education of Guangdong Province, Key Laboratory of Functional Molecules from Marine Microorganisms (Sun Yat-sen University), Guangzhou, Guangdong Province, China

& A unique serpin, kallistatin, displays vasodilatory, antiangiogenic, anti-inflammatory, and antioxidant activity. Difficulty and low efficacy of obtaining recombinant kallistatin limit the wide investigation of its biological and pathological function. The present study employed a codon optimization algorithm to redesign the kallistatin gene and achieved a high yield of recombinant kallistatin protein. The kallistatin codons were redesigned for a more suitable Escherichia coli host without altering amino acids. Base composition and GC% content were compared between synthetic optimized kallistatin (opti-kallistatin) and wild-type kallistatin (wt-kallistatin). Both opti-kallistatin and wt-kallistatin were purified using Ni-NTA His-binding resins through fast protein liquid chromatography (FPLC). The identity and purity of kallistatin were confirmed by Coomassie blue staining, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and Western blot analysis. The output of opti-kallistatin protein was 2-fold increase (2.09  0.23 mg=L) compared to wt-kallistatin (1.05  0.2 mg=L). These results suggest that more common codon optimization in the E. coli host significantly increases the yield of heterologous human protein yields. This approach will remarkably facilitate the further investigation of kallistatin in vitro and in vivo. Keywords angiogenesis, codon optimization, human KBP, Kallistatin, protein purification, serpin

Zhiyu Dai and Yifei Chen have contributed equally to this study. Address correspondence to Guoquan Gao, Department of Biochemistry, Zhongshan Medical School, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou 510080, China. E-mail: gaogq@ mail.sysu.edu.cn. Or contact Xia Yang. E-mail: [email protected]

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INTRODUCTION Kallistatin, termed human kallikrein-binding protein (HKBP), is a plasma glycoprotein belonged to the serine proteinase inhibitor (Serpin) super family. Kallistatin is first identified as a tissue kallikrein-binding protein, and strongly binds to tissue kallikrein.[1,2] Kallistatin is mainly synthesized and secreted by the liver, and is also found in a wide range of tissues like kidney, myocardium, and blood vessels.[3] Subsequently, kallistatin is proved to be a potent vasodilator and angiogenic inhibitor.[4–6] Furthermore, other functions such as anti-inflammatory and antioxidation properties have been investigated.[7–10] These findings suggest that kallistatin is a pleiotropic functional factor contributing to multiple biological and pathological processes. The kallistatin gene encodes 427 amino acids residues, including a 26-residue signal peptide and 401-residue mature peptide, with a calculated molecular mass of approximately 48.5 kD.[1,11] There are four potential glycosylation sites founded in translated kallistatin, which make the molecular mass of purified kallistatin from human serum about 58 kD.[11] Kallistatin shares 44–46% sequence homology with members of the Serpin family, such as human a1- antichymotrypsin, protein C inhibitor, a 1-antitrypsin, and rat kallikrein-binding protein.[11,12] The functional structure base of kallistatin has been identified in the hinge of the reactive center loop, P3 to P1 reactive site residues, and two putative heparinbinding regions, F helix and the loop between the H helix and C2 sheet.[11–15] Kallistatin is widely investigated as a multifunctional protein; large amounts of kallistatin protein are needed for functional and molecular mechanism studies in vitro and in vivo. Expression of a human gene in Escherichia coli is a common way to obtain high-scale biofunctional proteins, since that prokaryotic expression system has less cost and more convenient purification procedure than expressing protein in a human cell line. Although there are two reports about expression and purification of recombinant kallistatin in E. coli and Pichia pastoris, it is still limited by poor yields.[3,16] Unfortunately, heterogeneous genes are often difficult to express in another host. They may contain codons that the host rarely used, which is known as codon bias. There are 64 different codons but only 20 different translated amino acids, overabundant codons allow many amino acids to be encoded by more than one codon, and different organisms often show particular preferences for one of the several codons that encode the same amino acid.[17,18] Therefore, it is probable to maximize the likelihood of high protein expression by replacing the human codon with the more common bacterial codon in E. coli. In the present study we used the codon optimization algorithm to redesign the kallistatin codon without altering its amino acid sequence.[17–20]

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Moreover, codon adaptability, mRNA structure, and various cis elements in transcription and translation are critical to protein expression, which had been taken into consideration in our study. The present study provides useful strategy to obtain high-yield production of functional kallistatin. MATERIAL AND METHODS Downloaded by [Sun Yat-Sen University], [Guoquan Gao] at 23:09 09 December 2012

Materials The restriction enzymes BamHI and HindIII, Prime Star DNA polymerase, and T4 DNA ligase were purchased from Takara (Takara Bio, Inc., Shiga, Japan). DH5a strains were obtained from Takara (Takara Bio, Inc., Shiga, Japan). The pET28a vector, BL21(DE3) strain, and Ni-NTA His-Bind resin were purchased from Novagen (Madison, WI). All polymerase chain reaction (PCR) primers were synthesized by the Invitrogen. Basic fibroblast growth factor (bFGF) was sourced from R&D Systems (Minneapolis, MN). Cell Culture Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical vein cords of normal pregnancies obtained from the Department of Obstetrics and Gynecology (First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China) following a protocol described previously with some modifications.[21] These cells were cultured on flasks coated with 0.2% gelatin in serum-free media (SFM) supplemented with 15% fetal bovine serum (Life Technologies) and 30 mg=L endothelial cell growth supplement (ECGS), (Upstate Biotechnology, Lake Placid, NY, USA) in an atmosphere of 5% CO2 at 37 C. The medium was changed every 2 days until the cells reached confluence. Rabbit anti-human von Willebrand factor polyclonal primary antibody (Gene Tech, Shanghai, China) and FITC-labeled anti-rabbit immunoglobulin=FITC secondary antibody (Dako, Glostrup, Denmark) were used to identify HUVEC by an immunocytochemistry method (data not shown). To maintain uniform condition, all experiments on HUVECs were carried out between cell passages 4 and 6. Codon Optimization of Human Kallistatin cDNA for Expression in Escherichia coli The human full-length kallistatin DNA (1206 bp) was codon optimized for bacterial expression using the Genscript OptimumGene design platform, which employs a unique algorithm and proprietary codon usage table. Modifications were made throughout the sequence in approximately 22.5% of the DNA. Codon-optimized kallistatin (opti-kallistatin) was successfully

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assembled using overlapping polymerase chain reaction (PCR) (Genscript). The full-length DNA of wild-type kallistatin (wt-kallistatin) was amplified from the total RNA of human liver by reverse transcription-PCR as previously described.[22,23] The sense primer was ATGGATCCGATGGTGAGAGTTG CAG, and the antisense primer was GACAAGCTTTGGTTTCGTGGGGT.

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Construction of Plasmids for Expression of Opti-Kallistatin and wt-Kallistatin The synthetic opti-kallistatin and wt-kallistatin PCR product containing the mature kallistatin were cloned into the pET28a vector using the BamHI and HindIII restriction sites, respectively. The vector contains the N-terminal and C-terminal polyhistidine tag of the protein for Ni-NTA metal-affinity chromatography purification. The synthetic kallistatin plasmid construct opti-kallistatin=pET28a and wild-type kallistatin plasmid construct wt-kallistatin=pET28a were transformed to DH5a chemically competent cells. Several positive colonies were isolated from ampicillin agar plates and grown in Luria–Bertani (LB) medium containing 50 mg=mL ampicillin. Opti-kallistatin and wt-kallistatin plasmid DNAs were isolated and digested with BamHI and HindIII restriction enzymes, respectively, then subjected to agarose gels electrophoresis. Plasmids containing the DNA product size were subjected to sequencing (Invitrogen). Expression of Opti-Kallistatin and wt-Kallistatin The opti-kallistatin=pET28a and wt-kallistatin=pET28a recombinant vectors were transformed into 100ml BL21(DE3) competent cells for 30 min at 4 C, followed by 90 s at 42 C, then were incubated on ice for 5 min. Five hundred microliters of medium was supplemented for another incubation at 37 C for 45 min with constant shaking at 45 rpm. The medium containing cells was plated on ampicillin agar plates at 37 C overnight. BL21(DE3) strains containing the expression vector were grown in 20 mL LB medium containing 50 mg=mL ampicillin at 37 C for appropriately 3 hr; 1 mL of cells was collected and lysed by 1  SDS buffer as the before induction component, and then 1 mmol=L isopropyl-b-D-triogalactoside (IPTG) was added for an additional 4 hr or 8 hr at 37 C when OD600 ¼ 0.8. After 4 or 6 hr of growing, expressed proteins were subjected to SDS-PAGE and Western blot analysis. Purification of Kallistatin Protein The purification was carried out with Ni-NTA His-Bind resin by highperformance liquid chromatography (HPLC). The cells were harvested

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by centrifugation at 10,000  g for 30 min and completely resuspended with cold 10-mmol=L imidazole diluted by binding buffer (6.9 g=L NaH2PO4, 18 g=L NaCl, pH 8.0), then lysed by sonication. The supernatant fractions were collected by centrifugation at 15,000  g for 30 min, and filtered using a 0.45-mm filter (PALL). The supernatant was loaded onto a column filled with Ni-NTA binding resin (Novagen, Madison, WI), and then the flow was collected and reloaded two more times. Bound his-tagged wt-kallistatin and optikallistatin were eluted by elution buffer (40 mmol=L imidazole diluted by binding buffer) after washed by 300 mL washing buffer (20 mmol=L imidazole diluted by binding buffer). The purified kallistatin protein was identified using 12% SDS-PAGE and Western blot, and the purity of kallistatin was estimated by gray scanning using Image J software. For large-scale protein purification, cells were carried out using induction conditions of 1 mmol=L isopropyl b-D-1-thiogalactopyranoside (IPTG) at 37 C for 6 hr in 1 L LB medium containing 50 mg= mL ampicillin, then subjected to kallistatin purification. Then the purified protein was dialyzed for 12 hr twice using 10% glycerol diluted by phosphatebuffered saline (PBS). Finally, the endotoxin in recombinant proteins is removed to be less than 0.1 EU=mL by commercial ToxinEraser Endotoxin Removal kit (Genescript, Piscataway, NJ) according to the manual. Western Blot Analysis The cells were harvested by centrifugation and lysed for total protein extraction by 1  SDS buffer. Protein concentration was determined using the Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s protocol. Aliquots of equal amounts of protein (80 mg) from the lysate were subjected to Western blot analysis for the kallistatin using his-tag monoclonal antibody (Novagen, Madison, WI) as described previously.[24,25] Cell Viability Assay HUVECs were seeded at 1  104 cells=100 mL in gelatin-coated 96-well culture plates. After being starved overnight in serum-free M199 medium, the cells were treated with 20 ng=mL bFGF and 80nmol=L wt-kallistatin or opti-kallistatin or the same volume of sterile dialysate buffer at 37 C for 48 hr or 72 hr. The viable cells were quantified after 48 hr of incubation by CCK-8 assay (Dojindo Laboratories, Kumamoto, Japan) following the protocol recommended by the manufacturer. Statistical Analysis All data were expressed as mean standard deviation. SPSS 13.0 software was used for statistical evaluation using one-way analysis of variance

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(ANOVA) for comparison of more than two groups and using Student’s t-test for comparison of two groups. A p value less than 0.05 was considered significant.

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RESULTS Codon Comparison of Wild-Type Kallistatin and Optimized Kallistatin To enlarge the recombinant kallistatin outcome from E. coli, we reconsidered the codon bias usage in bacteria and optimized human wild-type kallistatin gene by replacing part of codons with bacterial more common codons according to the Gene Optimizer software algorithm. As shown in Figure 1, mature optimized kallistatin (opti-kallistatin) and mature wild-type kallistatin (wt-kallistatin) sequences were present, and there were substitutions of several human bias codons to those favored by bacteria without affecting translated amino acids. For example, human-favored codons were changed to bacterially favored ones, such as GAG to GAA (E), CCC to CCG (P), and AAG to AAA (K) (Figure 2). The codon frequency balance was also modulated to be more propitious for the bacteria host, for example, GUU (V), CAA (Q), and UGU (C) (Figure 2). Moreover, the base composition and GC% content were compared between wt-kallistatin and opti-kallistatin, and there was 3.4% less GC% content in opti-kallistatin than in wt-kallistatin (Figure 3), despite no amino acid sequence being changed. The codon adaption index (CAI), which represents the predicted expression level of a gene,[18] was upgraded from 0.64 to 0.91 after codon replacement.

Cloning and Expression of Wild-Type Kallistatin and Codon-Optimized Kallistatin Codon-optimized kallistatin was obtained using overlapping PCR, and the wild-type kallistatin was amplified using high-fidelity polymerase. Both wt-kallistatin gene and opti-kallistatin gene are inserted into pET28a vector with two 6  his-tag residues in the N terminal and C terminal of the multiple cloning site for convenient purification of the expression protein. PCR product gel assay showed that both wt-kallistatin and opti-kallistatin were successfully cloned into the pET28a vector, and the expected inserted DNA size is 1206 bp as shown in Figure 4A. These recombinant vectors were transformed into BL21(DE3); then kallistatin produced by E. coli was evaluated by Coomassie blue-stained SDS-PAGE, the results showed an apparent molecular mass of 50 kD protein after 4 hr and 8 hr of IPTG

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FIGURE 1 Sequence comparison between opti-kallistatin and wt-kallistatin genes: opti, codonoptimized (optimized codon are in gray color type); wt, wild type; aa, amino acid.

induction in both wt-kallistatin and opti-kallistatin transformed clones (Figure 4B).

Purification and Quantitative Yields of Wild-Type Kallistatin and Optimized Kallistatin Both proteins expressed by the wt-kallistatin and opti-kallistatin strain were purified by Ni-NTA His-binding resins using fast protein liquid

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FIGURE 2 Comparison of codon usage in bacteria, wt-kallistatin and opti-kallistatin. The figure shows the codon changes in optimized kallistatin and the frequency balance adjustments of codon usage between E. coli and opti-kallistatin.

chromatography (FPLC), and the purified protein showed an apparent band in the 50-kD molecular mass position by Coomassie blue-stained SDS-PAGE and confirmed by Western blot analysis using a His-tag antibody (Figure 4C), matching the calculated molecular weight from amino acid sequence. The purity of recombinant wt-kallistatin and opti-kallistatin was about 93% and 90%, estimated by gray scanning using Image J software. A large scale of protein expression was subjected to evaluate the yields of

FIGURE 3 Base composition and GC% content of wt-kallistatin and opti-kallistatin.

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FIGURE 4 Expression and purification of wt-kallistatin and opti-kallistatin in bacteria. (A) Agarose gel electrophoresis shows the inserted size (1206 bp) of wt-kallistatin and opti-kallistatin and the vector pET28a. (B) Coomassie blue stain SDS-PAGE analysis shows the expression levels of wt-kallistatin and opti-kallistatin induced by IPTG over 4 and 8 hr in BL21(DE3) strain. (C) Coomassie blue staining analysis showed the IPTG-induced and purified wt-kallistatin and opti-kallistatin. Lower panel, Western blot analysis confirmed both wt-kallistatin and opti-kallistatin using a his-tag monoclonal antibody.

wt-kallistatin and opti-kallistatin; we obtained 1.05  0.2 mg=L purified wt-kallstatin and 2.09  0.23 mg=L opti-kallistatin, respectively (Figure 5). In comparison, purified yield of opti-kallistatin expressed in BL21(DE3) represents 2-fold increase compared to wt-kallistatin (Figure 5).

Opti-Kallistatin Has Biological Activity Similar to wt-Kallistatin Kallistatin is well known as an endogenous potent angiogenic inhibitor.[4] To verify whether opti-kallistatin retains the same activity as wtkallistatin, we performed the CCK-8 assay to assess the effect of wt- and opti-kallistatin on activated endothelial cells. As shown in Figure 6, 20 ng=mL bFGF was used to induce HUVECs proliferation, since endothelial cell activation is essential for angiogenesis.[26] Here we showed that both wt- and opti-kallistatin recombinant proteins inhibited proliferating endothelial cell viability at 48 hr (wt 73.9  5.4%, opti 76.1  14.1%) and 72 hr (wt 65.4  5.3%, opti 67.8  8.6%), respectively. This finding suggests

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FIGURE 5 Yields of wt-kallistatin and opti-kallistatin. Quantitative measurement indicated 2-fold increase yield of opti-kallistatin protein over wt-kallistatin (n ¼ 3; asterisk indicates significant difference, p < 0.05).

FIGURE 6 Effect of wt-kallistatin and opti-kallistatin on cell viability. HUVECs were treated with 20 ng= mL bFGF and 80 nmol=L wt-kallistatin or opti-kallistatin for 48 hr or 72 hr. NC represent the group cell without bFGF treatment. The viable cells were quantified by CCK-8 assay. Data represent absorbance as percentages of respective control. Values statistically different from the control are indicated (n ¼ 8; asterisk indicates significant difference, p < 0.05).

that codon optimization approach improves outcome of biologically active kallistatin, and implies its potential significance for translational research.

DISCUSSION Kallistatin is widely produced in the human body, and presents multifunctional activities in biological process[27]; studies have demonstrated the importance of kallistatin for in vitro and in vivo models. The convenience and high-yield expression strategy of kallistatin are in great demand

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in investigating kallistatin’s profitable functions and mechanism. The present study was determined to optimize codon composition of human kallistatin in an E. coli host for high yields of recombinant kallistatin protein. Expression of human proteins in heterologous bacteria hosts plays a crucial role in the entire molecular biological industry, such as the industrial recombinant insulin and growth hormones.[28] Meanwhile, it is also very challenging since sometimes the human gene cloned is not expressed or yields less. Widely varied factors regulate and influence the gene expression level; however, a lot of studies have been conducted to improve heterologous expression, including optimizing host growth conditions and IPTG-induced concentration, or changing new E. coli strains.[17] Moreover, codons bias was taken into consideration since DNA sequence in one organism is often variant from other species.[20,29] The codon frequency of different organisms is different from each other,and the native gene employs tandem rare codons that can reduce the efficiency of translation or even disengage the translation machine.[30–32] Thus, the strategy of readjusting the exogenous recombinant protein codon into a host’s more common codon represents a profound advantage in heterologous protein expression. Here, we used a codon optimization algorithm to redesign the human kallistatin codon without altering amino acids sequence. We adjusted kallistatin codon by changing bacterial favored codons or balancing the codons frequencies (Figures 1 and 2), and upgrading the codon adaption index (CAI) from 0.64 to 0.91. In addition, GC content and an unfavorable peak were balanced to prolong the half-life of mRNA (Figure 3), and the stem–loop structures were broken to enhance ribosomal binding and mRNA stability. The pET system is robust for E. coli protein expression under T7 RNA polymerase, and it is convenient for easy purification of recombinant protein because of the N-terminal and=or C-terminal fusion histidine-tag.[33,34] We successfully cloned both wt-kallistatin and opti-kallistatin vectors (Figure 4A), and purified wt-kallistatin and opti-kallistatin proteins (Figures 4B and 4C), which was confirmed by SDS-PAGE and Western blot. The molecular mass of wt-kallistatin and opti-kallistatin on Coomassie blue staining gel in our study was approximately 50 kD, which is identical with the calculated molecular mass of kallistatin amino acids fused with the histidine tag. Our recombinant kallistatin has less molecular mass than the native kallistatin (58 kD); this may due to a lack of glycosylation in E. coli. However lack of glycosylation of kallistatin did not seem to affect the function of recombinant kallistatin (Figure 6); this phenomenon can also be observed for recombinant human pigment epithelium-derived factor (PEDF) and a1-antichymotrypsin produced in E. coli.[35,36] Furthermore, protein yields of opti-kallisatin are 2-fold increased compared to

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wt-kallisatin (Figure 5), suggesting that the increase in opti-kallistatin over wt-kallistatin expression may be due to the substitution of bacterial favored codons and by modulating the frequencies balance in E. coli host. Besides, codon optimization of kallistatin gene not only expressed a high level of protein, but exhibited the similar inhibition effect on activated endothelial cell proliferation. In conclusion, both wt-kallistatin and opti-kallistatin expression systems are successfully constructed, and heterologous kallistatin expression is greatly augmented by using the substitution of host favored codons without changing the native translational amino acids. Using a codon optimization strategy, a large amount of recombinant kallistatin could be easily accessed for further exploration on evaluating the anti-inflammatory and antiangiogenic activities of kallistatin in cell and animal models.

ACKNOWLEDGMENTS This study was supported by the National Nature Science Foundation of China, grant numbers 30600724, 30700120, 30872980, 30971208,30973449, 81070746, and 81001014; National KeySci-Tech Special Project of China, grant number 2009ZX09103-642; Program for Doctoral Station in University, China, grant number 20100171110049; Key Project of Nature Science Foundation of Guangdong Province, China, grant number 10251008901000009; Program for Chang Jiang Scholars and Innovative Research Team in University, China, grant number PCSIRT0947; Guangdong Natural Science Fund, China, grant number 10151008901000007; Key Sci-tech Research Project of Guangdong Province, China, grant number 2008B080703027; Key Sci-tech Research Project of Guangzhou Municipality, China, grant number 2008Z1-E231; and Program for Young Teacher in University, China, grant numbers 09YKPY73 and 10YKPY28.

REFERENCES 1. Wang, M.Y.; Day, J.; Chao, L.; Chao, J. Human Kallistatin, a New Tissue Kallikrein-Binding Protein: Purification and Characterization. Adv. Exp. Med. Biol. 1989, 247B, 1–8. 2. Chao, J.; Tillman, D.M.; Wang, M.Y.; Margolius, H.S.; Chao, L. Identification of a new TissueKallikrein-Binding Protein. Biochem. J. 1986, 239(2), 325–331. 3. Chai, K.X.; Chen, L.M.; Chao, J.; Chao, L. Kallistatin: A Novel Human Serine Proteinase Inhibitor. Molecular Cloning, Tissue Distribution, and Expression in Escherichia coli. J. Biol. Chem. 1993, 268(32), 24498–24505. 4. Miao, R.Q.; Agata, J.; Chao, L.; Chao, J. Kallistatin Is a New Inhibitor of Angiogenesis and Tumor Growth. Blood 2002, 100(9), 3245–3252. 5. Chao, J.; Miao, R.Q.; Chen, V.; Chen, L.M.; Chao, L. Novel Roles of Kallistatin, a specific Tissue Kallikrein Inhibitor, in Vascular Remodeling. Biol. Chem. 2001, 382(1), 15–21.

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