Expression and characterization of functional human vascular endothelial growth factor (VEGF165) in Kluyveromyces lactis

Among the vascular endothelial growth factor (VEGF) family variants, the 165-amino acid isoform (VEGF 165 ) is the best characterized and most potent endothelial cell mitogenic factor. It is known that VEGF 165 mediates angiogenesis and has the potential for the therapeutic applications. In this study, the expression system of Kluyveromyces lactis that produces the recombinant human VEGF 165 has been evaluated. The gene encoding human VEGF 165 was successfully cloned in the pKLAC2 expression vector containing a strong LAC4 promoter, after which a pKLAC2-VEGF 165 plasmid was constructed. After the transformation, the recombinant human vascular endothelial growth factor 165 (rhVEGF 165 ) was expressed in K. lactis GG799 cells (~ 5.7 mg/L) conrmed by SDS-PAGE and Western blotting and downstream purication processed comprising ammonium sulphate precipitation and anity chromatography. The biological activity of the puried rhVEGF 165 was conrmed by the proliferation of the human umbilical vein-derived endothelial cells (HUVEC) in a dose- and time-dependent manner. The K. lactis-derived rhVEGF 165 exhibited a higher proliferative activity compared with a commercially available rhVEGF 165 with a half-maximal effective concentration of 3.0. Cell migration analysis was conducted to evaluate the in vitro wound healing effect of the produced rhVEGF 165 via a scratch assay. These ndings indicate that K. lactis could be a suitable host for secreting bioactive human VEGF 165 for therapeutic use.


Introduction
Vascular endothelial growth factor (VEGF) is a family of protein that requires speci c mitogen for the growth of endothelial cells (Leung et al. 1989). It plays a signi cant role in fundamental processes, such as growth of new capillaries from the existing vasculature vessels (angiogenesis) and formation of blood vessels from endothelial precursor cells (vasculogenesis) (Ferrara and Kerbel 2005). Originally, VEGF was identi ed as a vascular permeability factor (VPF) (Senger et al. 1983;Dvorak et al. 2006) and then Ferrara and Henzel (1989) named as VEGF.
The VEGF family belongs to the platelet-derived growth factor (PDGF) and comprises of the following: VEGF-A, placental growth factor, VEGF-B, VEGF-C, VEGF-D, VEGF-E and VEGF-F (Li and Eriksson 2001;Shibuya 2003). VEGF-A, which is generally referred to as VEGF, is the founding member of the family; it characterizes eight exons. Due to the alternative splicing of exons 6 and 7 of VEGF pre-mRNA, various isoforms are generated, such as VEGF 121 , VEGF 121b , VEGF 145 , VEGF 165 , VEGF 165b , VEGF 189 and VEGF 206 (Tischer et al. 1991;Ferrara 2004;Holmes and Zachary 2005). All these isoforms have a common amino terminal domain consisting of 110 amino acids, while the length of carboxyl-terminal differs.
VEGF 165 is the rst VEGF splice variant of the VEGF-A family that has been described. It has a molecular weight of 38.2 kDa and a theoretical pI of 8.6 (Keck et al. 1997). The disulphide-linked homodimeric protein VEGF 165 is the most abundant and best-characterized isoform that is comprised of two 165 amino acid polypeptide chain monomers (Ferrara 1991). Each of the two monomers has a single glycosylation site and a cystine-knot motif. VEGF 165 digestion by plasmin yields two domains: amino terminal homodimer (amino acids 1-110), which contains the VEGF receptor binding site for the kinase domain receptor (KDR), and the fms-like tyrosine kinase receptor (Flt-1). The carboxyl-terminal portion of the VEGF 165 (amino acids 111-165) is known as the carboxyl-terminal heparin-binding domain; it mediates heparin a nity, which is important for the mitogenic effect on the endothelial cells (Keyt et al. 1996;Takahashi and Shibuya 2005). Numerous different cells and tissues are known to express VEGF 165 as a strong mitogenic factor under physiological and pathological conditions (Lauer et al. 2002).
VEGF mainly contributes to vascular development through angiogenesis by inducing endothelial cell proliferation and migration. Moreover, it regulates these functions by binding to speci c tyrosine kinase VEGF receptors (VEGFRs) on the surface of vascular endothelial cells. The activation of VEGF-A with VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk1 in mice) is necessary for angiogenesis and vascular permeability under both physiological and pathological conditions. Conversely, at the early stage of embryogenesis and in the development of lymphatic vessels, VEGF-C/D and its receptor VEGFR-3 mainly regulate angiogenesis (Shibuya 1995;Shibuya 2006;Alitalo and Carmeliet 2002).
The function of VEGF is not limited to angiogenesis; VEGF also plays a signi cant role in normal physiological functions, such as wound healing, menstrual cycle, bone formation, haematopoiesis and neural development (Gerber et al. 1999;Ferrara et al. 1996;Chintalgattu et al. 2003;Reichardt and Tomaselli 1991). Moreover, it can be released in high amounts by activated platelets and increase the formation of granulation tissue at the wound site (Hoeben et al. 2004;Dvorak et al. 1995). Under stress conditions, such as hypoxia, chemotherapy and radiotherapy, VEGF acts as a survival factor secreted by tumour cells (Riedel et al. 2004;Gorski et al. 1999, Scott et al. 1998). The formation of new vessels causes tumour cells to enter the circulation and increase metastasis. Due to its role in tumour formation and survival strategies, VEGF is considered to be an attractive target for anti-angiogenic medication (Gardner et al. 2007). VEGF and its receptors are required for embryonic angiogenesis and the development of vascular network , Tammela et al. 2005. In normal adult health, VEGF plays a signi cant role in non-malignant diseases, such as rheumatoid arthritis (Lee et al. 2001), psoriasis (Xia et al. 2003), diabetes and ischemic retinopathies (Aiello et al. 1995).
VEGF is synthesized by numerous cell types, including endothelial cells, tumour cells, broblasts, platelets, macrophages, neutrophils, keratinocytes, T cells and renal mesangial cells (Boocock et al. 1995;Sunderkotter et al. 1994, Verheul et al. 1997Frank et al. 1995;Iijima et al. 1993). Aside from bacterial expression (Pizarro et al. 2010;Nguyen et al. 2016), several eukaryotic expression host systems have been employed for the recombinant production of VEGF, including yeast (Mohanraj et al. 1995;Kang et al. 2013), Chinese hamster ovary cells (CHOs) (Lee et al. 2008), insect cells (Lee et al. 2006), transgenic rice (Chung et al. 2014) and silkworm (Wu et al. 2004). The most preferred system is Escherichia coli (E. coli); however, it exhibits drawbacks, such as the tendency to form inclusion bodies, protein misfolding and di cult puri cation steps (Nguyen et al. 2016). In addition, yeast and bacteria are cost-effective systems, unlike other higher organisms, such as insects or mammalian cells; however, unlike bacteria, yeasts do not require refolding for activity.
Kluyveromyces lactis (K. lactis) is an emerging yeast host for the production of heterologous protein. It has the ability to obtain high levels of protein, thus making it particularly suitable for industrial applications. In addition to being used as a host for protein expression, K. lactis has potential for numerous biotechnological applications, for example, as an infant formula component, protein supplement, avour enhancer, commercial enzyme producer, lactase and lactic acid source and probiotic single-cell protein (Bonekamp and Oosterom 1994;Belem and Lee 1998;Oishi et al. 1999;Colussi et al. 2005;Porro et al. 1999;Ghaly et al. 2005).
Moreover, K. lactis has all the advantages of other yeast expression systems, such as the following: it has the ability to obtain high culture densities and high yield; it exhibits posttranslational modi cation mechanisms similar to mammals; it has the ability to form disulphide linkages; and it can be used for scalable fermentation and for the production of endotoxins and carcinogens (Kim and Kim 2016). The genome of K. lactis is completely sequenced; thus, it can be easily manipulated (Dujon et al. 2004). It is also possible to use both integrative and episomal expression vectors for the K. lactis expression. The status generally recognized as safe (GRAS), a designation provided by the US Food and Drug Administration, makes K. lactis particularly safe and useful in food and feed applications. For enzymes and proteins expressed from K. lactis, endotoxin analysis is not required (Bonekamp and Oosterom 1994). A standard inexpensive culture medium is su cient for the rapid growth of K. lactis, and highmolecular-weight proteins can be secreted in the culture medium. For industrial applications, the induction of recombinant protein in K. lactis is important as it has constitutive promoters and also does not require expensive or highly ammable chemicals, such as methanol as in P. pastoris. Therefore, the use of explosion-proof equipment is not necessary (Rocha et al. 2011;Rosa et al. 2013).
In K. lactis, approximately 100 heterologous proteins have been successfully produced and many of these proteins are commercially available in different industries (Spohner et al. 2016). The achievement of the use of K. lactis as a host for protein expression in the food industry has continued in the largescale production of therapeutic proteins in the pharmaceutical industry. Numerous pharmaceutical proteins, such as interleukin 1-β, insulin precursor, interferon-α, β-lactoglobulin, macrophage colonystimulating factor (M-CSF) and various antibodies, have been produced in K. lactis (Fleer et al. 1991;Feng et al. 1997;Chen et al. 1992;Rocha et al. 1996;Hua et al. 1994;Swennen et al. 2002).
In this study, the gene of the human VEGF 165 was inserted into the K. lactis pKLAC2 expression vector and transformed into the K. lactis GG799 yeast cells. The secreted rhVEGF 165 was puri ed from the culture medium via a nity chromatography. In addition, analysis of the bioactivity of the produced VEGF 165 was conducted by testing in vitro for proliferation and wound healing assay.

Materials And Methods
Strains, plasmids and growth conditions pET22b plasmid containing the human VEGF 165 cDNA (GenBank accession no. AF486837.1) was obtained from Biomatik (Biomatik Corporation, Cambridge, Canada). E. coli DH5a was utilized for the maintenance and manipulation of plasmids. The K. lactis Protein Expression Kit (New England BioLabs, Massachusetts, ABD) containing the pKLAC2 expression vector was utilized. Moreover, the E. coli DH5a cells were grown in the Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl and pH 7.0) at a temperature of 37 °C. The K. lactis GG799 cells were grown in the YPGlu medium (1% yeast extract, 2% peptone and 2% glucose) at 30 °C in a shaker at 200 rpm. In addition, expression of the transformed K. lactis GG799 cells was performed in the YPGal medium (1% yeast extract, 2% peptone and 4% galactose). All the chemicals were purchased from Sigma-Aldrich (Darmstadt, Germany) and Merck (Merck Millipore, Darmstadt, Germany). rpm. The genomic DNA of yeast cells was isolated according to the method described by Harju et al. (2004). The positive transformant containing the VEGF 165 sequence was screened by PCR using specific primers (used before for cloning) and then analysed by agarose gel electrophoresis. In addition, the positive transformants were grown in the YPGal medium (1% yeast extract, 2% peptone, 4% galactose), and the protein expression levels of the culture medium samples were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine the best clone that secreted the VEGF 165 . The recombinant K. lactis clone in the YPGal medium was stored by adding 60% (v/v) glycerol and freezing at −80 °C.
Expression of recombinant human VEGF 165 in K. lactis GG799 The starter culture of the VEGF 165 producer K. lactis GG799 clone was grown for 2 days at 30 °C in 5 ml YCB medium containing 5 mM acetamide. The growth culture was inoculated at a ratio of 1:100 into 300 ml YPGal medium (4% galactose) and was allowed to grow at 30 °C for 3 days. Cell harvesting was performed by centrifugation at 6,000 × g for 45 min after incubation.
Puri cation ofhuman VEGF 165 The cell-free supernatant of the transformed K. lactis containing secreted recombinant human VEGF 165 were precipitated with 60% ammonium sulphate solution in an ice bath for 60 min and then centrifuged at 10,000 × g for 20 min. Then, the protein pellet was resuspended in 100 mM phosphate buffer (pH 7.5) and was subjected to a nity chromatography for puri cation through the binding of the hexahistidine tag (located at the C-terminal end of the VEGF 165 ) using a 0.5 ml nickel-nitrilotriacetic acid (Ni-NTA) resin column (Qiagen, Hilden, Germany). To remove non-speci cally bound impurities, the column was washed with phosphate buffer. The VEGF 165 was eluted with phosphate buffer containing 300 mM imidazole. The eluted protein was then transferred to the dialysis tubing containing 1000 kDa MWCO (Spectrum Laboratories, California, USA) and dialysed against 20 mM HEPES (pH 7.4) at 4 °C. The protein concentration was quanti ed via absorbance spectroscopy at 280 nm using an extinction coe cient of 24,950 M −1 cm −1 .

Western blot analysis
The protein samples were subjected to electrophoresis under the denaturing conditions using 12% SDS gel stained with Coomassie Brilliant Blue R-250, as described by Laemmli (1970). The protein bands on the gel were transferred onto the polyvinylidene di uoride (PVDF) membrane for Western blot analysis. The membrane was blocked with 5% (w/v) skim milk in phosphate-buffered saline (PBS) for 30 min at room temperature. The membrane blot was washed and rinsed with PBS and incubated with 6xHis-Tag (C-term)/AP Monoclonal Antibody (Invitrogen, Carlsbad, CA, USA) at 1:3000 dilution for 2 h at room temperature. After washing with blocking buffer for three times, the membrane was incubated with 5bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate solutions (BCIP/NBT) (Sigma-Aldrich, Darmstadt, Germany) in colour development buffer. Germany) supplemented with 15% heat-inactivated fetal bovine serum (FBS), 1% L-glutamine and 0.1% gentamicin sulphate at 37 °C in a 5% CO 2 humidi ed atmosphere. Assay for cell proliferation was performed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to evaluate the biological activity of the produced VEGF 165 . Then, HUVECs were seeded in 96-well plates at a density of 4 × 10 3 per well and treated with the purified VEGF 165 and the commercial E. coli-derived VEGF 165 (Abcam, Cambridge, UK) at various concentrations for 2 and 6 days. The untreated cells were incubated as a negative control to determine the relative number of the treated cells. Three replicates for each concentration were performed. After cell culture, the cells were incubated with MTT (5 mg/mL) for 4 h. The culture media were removed, and the obtained formazan crystals were solubilized in dimethyl sulfoxide. The absorbance of each well was read at 570 nm using a microplate reader. Experiments were repeated in triplicate and expressed as mean ± standard error (SE). The group means were compared using Duncan's test or a one-way analysis of variance (ANOVA). A p value < 0.05 was considered as statistically significant.
In vitro scratch wound assay Scratch assay was performed to determine the effect of VEGF 165 on wound healing. L929 mouse broblast cells were seeded in a 6-well plate (10 4 cells/well) in the DMEM culture medium and grown to con uence. Then con uent cell layers were scratched with a pipette tip, and cellular debris was removed by washing off with PBS. The cells were treated with DMEM with 15% FBS containing 5 and 10 ng/mL of E. coli-derived and K. lactis-derived VEGF 165 . The controls received only fresh DMEM. The cells were incubated at 37 °C in a humidi ed 5% CO 2 atmosphere. Then, an image of the scratch area was captured using a phase-contrast inverted microscope (4X magni cation, Olympus CKX41), and the percentage of the scratch area was analysed using an Olympus analysis software.

Results And Discussion
Construction of the pKLAC2-VEGF 165 expression vector To achieve protein secretion, the VEGF 165 gene was cloned downstream of the K. lactis α-MF secretion domain, which resulted in the expression of an α-MF fusion protein. The human VEGF 165 gene was ampli ed from the pET22b-VEGF 165 vector using gene-speci c primers. Gradient PCR was performed to optimize the annealing temperature (65 °C) (Additional le 1: Figure S1). The ampli ed VEGF 165 and pKLAC2 plasmid DNA were separately digested with restriction enzymes (XhoI and EcoRI) and ligated with each other to generate pKLAC2-VEGF 165 (Fig. 1A). E. coli DH5a cells were transformed with the resulting recombinant plasmid and incubated on LB agar medium, including ampicillin (100 mg/mL). A total of 12 single colonies were randomly selected. The plasmid DNA of the clones analysed by PCR and three of these clones revealed the PCR product of the 633 bp VEGF 165 . Based on the results of the restriction enzyme digestion with NcoI-EcoRI and HindIII-EcoRI enzyme couples, only one clone (clone 2) was used for the transformation into K. lactis (Additional le 1: Figure S2).

Expression of VEGF 165 in K. lactis
The SacII-linearized plasmid pKLAC2-VEGF 165 was transformed into competent K. lactis GG799 cells via electroporation. The expression cassette was targeted for insertion into the LAC4 promoter region of the K. lactis chromosome by the 3' and 5' ends of the P LAC4-PBI promoter by homologous recombination.
pKLAC2 contains a variant of the strong K. lactis lactase LAC4 promoter, which is located in the vector pKLAC2 and acetamidase gene (amdS) for the positive selection of the transformed strains in a nitrogenfree medium. This medium contains acetamide as the only nitrogen source. A total of 14 transformants were randomly selected, and integration of the expression cassette into the K. lactis genome was confirmed by genomic DNA PCR, with the use of speci c primers of the VEGF 165 gene (Fig. 1C). The VEGF 165 gene (633 bp) was observed in 12 of these transformants (except for clone 2 and clone 4 in Fig.   1C). VEGF 165 secretion was evaluated using small-scale expression assays in 5 mL YCB medium containing 5 mM acetamide. Then, SDS-PAGE analysis of the culture supernatant was conducted to con rm the presence of VEGF 165 . Four positive transformants that successfully secreted the target protein were analysed by nucleotide sequencing to con rm the integration of the recombinant expression cassette into the K. lactis GG799 chromosome (Additional le 1: Figure S3).
The positive clone that exhibits the highest homology found in the GenBank database was grown in 300 ml YPGal medium. The VEGF 165 was expressed-fused with α-MF secretion domain and processed in the Golgi complex. Subsequently, it was secreted in the fermentation medium. The fusion proteins were cleaved by Kex protease at the processing site (KR↓) between the α-MF domain and N-terminus of the VEGF 165 gene. The supernatant of the medium was precipitated, and then, the precipitant solved in phosphate buffer was loaded to Nickel a nity chromatography. The eluted recombinant human VEGF 165 was subjected to 12% (w/v) SDS-PAGE under reducing conditions and visualized by Coomassie blue staining. The non-glycosylated recombinant human VEGF 165 monomer has a MW of approximately 20 kDa as reported in previous studies (Fiebich et al. 1993;Catena et al. 2010;Peretz et al. 1992). As presented in Fig. 2, the MW of VEGF 165 was approximately 30 kDa as a result of adding tags (restriction enzyme sites and His-tag). Moreover, VEGF 165 glycosylation varies according to the host organism and the effects of the MW of the protein. It was reported that the CHO-derived VEGF 165 exhibits higher MW compared with the E. coli-derived and insect cell-derived VEGF 165 due to different glycosylation mechanisms (Lee et al. 2008). The yield of the produced recombinant human VEGF 165 was determined by measuring the absorbance at 280 nm and using an extinction coefficient of 24950 M -1 cm -1 . The highest expression level of the analysed clones was 5.7 mg/L of VEGF 165 . It has been reported that recombinant human VEGF 165 is expressed in various expression systems, such as E. coli at 1.5 mg/L (Taktak-BenAmar et al. 2017) and Saccharomyces cerevisiae at 4 mg/L (Kang et al. 2013). In other studies, the expression levels of 80 mg/L (Lee et al. 2008) for CHO cell cultures and 20 mg/L (Lee et al. 2006) for insect cells at were achieved. The yields in previous studies related recombinant heterologous protein production such as human IFN-γ (2 mg/L) and hepatitis C virus E2 glycoprotein (1 mg/L) are comparable to the results of this study (Spohner et al. 2016).
Biological activity of the produced VEGF 165 To con rm the biological activity of the hVEGF 165 protein expressed and puri ed in this study, cell proliferation assay was performed by MTT assay. The growth of the HUVECs was stimulated by rhVEGF 165 in a dose-dependent manner. As presented in Fig. 3A, recombinant human VEGF 165 signi cantly enhanced the proliferation of HUVEC cells for 2 days (p < 0.0001). The results revealed that VEGF 165 produced in K. lactis exhibits higher mitogenic activity than the commercial E. coli-derived human VEGF 165 for all VEGF 165 concentrations (Additional le 1: Table S1). Particularly, 2.5 ng/mL of VEGF 165 exhibited the greatest proliferative activity (2.83% cell viability) (p < 0.001), which was higher than that of the commercial VEGF 165 (2.64%) (Additional le 1: Table S2). Accordingly, this concentration was employed in the subsequent time-dependent assay. The proliferative effect for 2 and 6 days was compared with absorption at 570 nm. After 6 days, the rate of proliferation was increased, and the produced VEGF 165 exhibited higher proliferation in the absorbance of 0.91 compared with the commercial VEGF 165 (0.85). In general, the half-maximal effective concentration (EC 50 ) of commercial rhVEGF 165 has been reported as 1-6 ng/mL. In this study, the EC 50 of the K. lactis-derived rhVEGF 165 protein was calculated to be approximately 3.0 ng/mL, which means a similar bioactivity to previous studies in CHO cells and insect cells (Lee et al. 2008;Lee et al. 2006). L929 mouse broblast cells treated with VEGF 165 were utilized in the wound healing assay. The VEGF 165 was found to facilitate wound healing at 5 and 10 ng/mL concentrations (Fig. 4) compared with the untreated control. Moreover, it has a similar effect to the commercial E. coli-derived VEGF 165 (Additional le 1: Table S3). It was clearly observed that, after 24 and 48 h of incubation at a concentration of 10 ng/mL, the percentages of the wound site treated with K. lactis-derived VEGF 165 were 69,54% and 85,71%, respectively. Meanwhile, the percentages of the cell-covered area were 73,48% and 93,12% for cells treated with E. coli-derived VEGF 165 and 55,24% and 78,59% for untreated cells. The migration assay con rmed that VEGF 165 is biologically active and that the ability of L929 mouse broblast cells to heal wound was promoted by VEGF 165. These results indicated that the proliferation e ciency of K. lactisderived VEGF 165 on HUVECs was higher than that of the commercial E. coli-derived VEGF 165 . Contrarily, the produced VEGF 165 in uenced the migration of L929 mouse broblast cells at a slower rate compared with E. coli-derived VEGF 165.

Conclusion
In this study, the expression of biologically active rhVEGF 165 protein in the K. lactis expression system, which has been used extensively as a host for heterologous protein expression, was successfully established. The recombinant human VEGF 165 was expressed in K. lactis GG799 cells and secreted in the culture medium. The VEGF 165 was puri ed functionally active from K. lactis culture medium by ammonium sulphate precipitation, Ni-a nity chromatography and dialysis. The produced K. lactisderived VEGF 165 exhibited higher biological activity than the commercially available E. coli-derived VEGF 165 . Moreover, it stimulated the proliferation of HUVECs. However, the migration effect of the rhVEGF 165 was tested on L929 mouse broblast cells. The addition of rhVEGF 165 to cell culture media led to a signi cant increase in migration, which contributed to an accelerated rate of wound healing in an in vitro scratch wound model when compared with untreated controls. In total, these observations indicate that rhVEGF 165 can be exploited as a therapeutic agent for the basic investigation of physiological states, such as wound healing and several diseases related to VEGF 165 . Abbreviations VEGF, vascular endothelial growth factor; VEGF 165 , vascular endothelial growth factor 165; rhVEGF 165 , recombinant human vascular endothelial growth factor 165; K. lactis, Kluyveromyces lactis; HUVEC, human umbilical vein derived endothelial cells; PDGF, platelet-derived growth factor; KDR, kinase domain receptor; Flt-1, fms-like tyrosine kinase receptor; VEGFRs, VEGF receptors; CHOs, Chinese hamster ovary cells; E. coli, Escherichia coli; GRAS, generally recognized as safe; M-CSF, macrophage colony-stimulating factor; LB, Luria-Bertani; PCR, polymerase chain reaction; a-MF, a-mating factor; YCB, yeast carbon base; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; Ni-NTA, nickel-nitrilotriacetic acid; PVDF, polyvinylidene di uoride; BCIP/NBT, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate solutions; DMEM, Dulbecco's Modi ed Eagle's Medium; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ANOVA, one-way analysis of variance; EC 50 , half-maximal effective concentration.

Declarations
Ethics approval and consent to participate All the authors have read and agreed to the ethics for publishing the manuscript. Construction of the pKLAC2-VEGF165 expression vector and integration into the K. lactis genome. a) Schematic diagram of the VEGF165 recombinant expression vector based on the pKLAC2. b) Genomic integration of linear pKLAC2-VEGF165 expression cassette into the K. lactis genome. c) Confirmation of the integration of the expression cassettes into the K. lactis genome by PCR. The numbers correspond to the randomly selected K. lactis clones and "c" to the amplification product using pET22b-VEGF165 plasmid as a positive control.

Figure 2
Puri cation of recombinant 6XHis-tagged VEGF165 from K. lactis. a) Schematic diagram of the puri cation process. b) SDS-PAGE (12% Tris-glycine gel) analysis of puri ed fraction recombinant human VEGF165. M, molecular-weight marker in kDa. The spent culture medium of untransformed K. lactis strain GG799 (negative control) was analysed after ammonium sulphate precipitation (Lane 1) and a nity chromatography (Lane 3). The recombinant human VEGF165 in the spent culture medium of transformed K. lactis strain GG799 with pKLAC2-VEGF165 expression cassette following ammonium sulphate precipitation (Lane 2) and eluted VEGF165 following a nity chromatography (Lane 4). c) Western blot analysis of puri ed VEGF165 with an anti-His antibody.

Figure 3
The dose-and time-dependent effects of the produced VEGF165 and commercially available VEGF165 on the proliferative activity of HUVECs. MTT assay was performed. a) In a dose-dependent test, HUVECs were seeded and incubated at 37 °C for 5 days with various VEGF165 concentrations. b) In a timedependent test, HUVECs were seeded and incubated at 37 °C for 2 or 6 days with 2.5 ng/mL of VEGF165.
The results are presented as cell viability (%) and absorbance (570 nm).