摘要
谷氨酸特异性内肽酶（GSE）是一种对谷氨酸羧基形成的肽键具有很强专一性的丝氨酸蛋白酶，在蛋白质序列及结构分析、多肽合成及活性多肽回收方面有着广泛的应用。由于大多数谷氨酸特异性内肽酶的晶体结构目前无法获得，因此我们对于其底物特异性的机制还不够了解。地衣芽孢杆菌谷氨酸特异性内肽酶（GSE-BL）与应用较广泛的来自金黄色葡萄球菌V8菌株的谷氨酸特异性内肽酶（V8-GSE）相比，对谷氨酸有着更强的底物专一性和更高的催化效率。因此，对GSE-BL进行表达纯化，并对其结构及功能进行深入分析对于了解其作用机制及拓展谷氨酸特异性内肽酶的应用范围有着十分重要的意义。
重组GSE-BL前体在大肠杆菌胞内以无活性的包涵体形式表达，经过透析复性及胰酶处理后，我们得到有活性的成熟GSE-BL，其产量为140.5mg/L。Western blot鉴定、酶活力测定及氨基酸序列测定结果表明其复性过程中自切割形成中间体，其成熟化过程是逐步进行的。GSE-BL前体第-1位的赖氨酸突变成为谷氨酸后，可以在不引入外源胰酶的情况下得到成熟GSE-BL，从而为制备有活性的成熟提供了一种新方法。但突变后的GSE-BL对于Z-Phe-Leu-Glu-pNA的活性降低了27.4%，表明第-1位置的Lys突变成为Glu对GSE-BL的正确折叠有一定程度的影响。不包含前肽片段的GSE-BL在大肠杆菌中主要以可溶形式表达，相应纯化产物对Z-Phe-Leu-Glu-pNA几乎没有酶活力；27kDGSE-BL中间体主要以包涵体形式表达，相应成熟化产物对Z-Phe-Leu-Glu-pNA几乎没有酶活力，从而证明前肽的存在对于GSE-BL正确折叠形成有活性的构象是十分必要的。
对GSE-BL的酶学性质研究结果表明，GSE-BL的最适反应pH为8.5，最适反应温度为37℃，对Z-Phe-Leu-Glu-pNA的Km值为1.495±0.034mM，最大反应速率Vmax为50.237μmol/mg.min。2mM钙离子的存在能显著提高重组成熟GSE-BL在50℃的热稳定性，EDTA能部分抑制成熟GSE-BL的酶活力。GSE-BL对于Z-Phe-Leu-Glu-pNA的催化效率是对于Z-Asp-pNA的催化效率的925倍。
我们以已知晶体结构的中间芽孢杆菌谷氨酸特异性内肽酶（GSE-BI）为模板，对GSE-BL进行了同源建模，预测GSE-BL中存在由His47、Asp96和Ser167组成的通过质子传递形成的稳定的催化活性中心，并用点突变实验分别进行了验证。我们还通过点突变实验证明了Val2、Cys32、Arg89和His190对于GSE-BL催化活性中心的形成和其底物特异性的重要性。其中Val2参与底物结合口袋的形成，对于GSE-BL的正确折叠十分重要；Cys32与底物结合，且参与二硫键的形成；Arg89为底物结合口袋提供正电荷，方便底物进入口袋；而His190与Glu残基的γ-羧基结合，对谷氨酸的负电荷进行补偿。Cys181突变成为Ala会降低GSE-BL的热稳定性。Phe57突变成为Ala能使GSE-BL对于Z-Phe-Leu-Glu-pNA的催化效率提高50%。Asp51Arg、Glu101Ala和Glu104Arg的突变影响GSE-BL催化域的电荷分布，导致GSE-BL无法形成稳定的催化活性中心。
本论文将GSE-BL应用于重组蛋白标签切除和活性肽的成熟化。
透明颤菌血红蛋白（VHb）是一种能提高生物体对氧气利用率的血红蛋白。VHb在大肠杆菌内重组表达，GSE-BL酶切15min可去除91.6%VHb-His6的组氨酸标签，从而获得与天然VHb氨基酸序列一致的VHb。无标签VHb的产量为31.8mg/L。去除标签的VHb对CO的亲和力比VHb-His6更高。无标签VHb对过氧化氢的最大反应速率为1169μmol/g.min，而VHb-His6对过氧化氢的最大反应速率为781μmol/g.min。0.1mM无标签VHb添加到产碱杆菌ATCC31555发酵液中能使韦兰胶的产量提高16.2%，而添加0.1mM VHb-His6仅使韦兰胶产量提高4.9%。
人胰高血糖素是一种能促进肝糖原分解从而提高人体内血糖含量的活性多肽。我们在毕赤酵母菌株中重组表达了人胰高血糖素，重组人胰高血糖分泌到胞外上清液中，纯化的重组人胰高血糖素用GSE-BL处理1h能去除91.6%重组胰高血糖素的组氨酸标签，得到与人胰高血糖素标准品分子量大小一致及HPLC保留时间一致的短肽。去标签的胰高血糖素的产量为50.5mg/L。
人β防御素-2（HBD-2）是一种富含半胱氨酸的小分子阳离子抗菌肽。我们在大肠杆菌内对HBD-2前原肽进行了重组表达，在成熟HBD-2序列前引入Glu残基作为GSE-BL的识别位点。纯化后的HBD-2前原肽经GSE-BL处理1h得到成熟的HBD-2，其产量为23.2mg/L。成熟HBD-2对于铜绿假单胞菌的抑菌能力强于对金黄色葡萄球菌的抑菌能力。成熟HBD-2对人结肠癌细胞HCT-8的毒性要强于对小鼠骨髓来源的DC细胞的毒性。
Glutamate-specific endopeptidase (GSE) is a kind of serine protease, which have strongsepcificity for peptide bonds formed by carboxyl of Glu residues，and GSEs have a broadapplication in the field of protein sequence and structure analysis, peptide synthesis and therecovery of bioactive peptide. The specifity of GSEs is not thoroughly understood until nowbecause the crystal structure of most GSEs have not been resolved. Compared with Glutamatespecific endopeptidase from Staphylococcus aureus V8(V8-GSE) which have been widelyused in many industries, Glutamate-specific endopeptidase from Bacillus lichefomis (GSE-BL)exhibits higher catalytic efficiency and better specificity towards Glu residues. Hence, it is ofgreat significance for widening the application of GSEs and understanding the mechanism ofaction of GSEs to investigate the structure and function of GSE-BL.
Recombinant GSE-BL was expressed in E.coli as inative inclusion bodies, mature activeGSE-BL was obtained with a yield of140.5mg/L after dialysis refolding, and trypsintreatment. The results of Western blot, enzymatic activity assaying and amino acid sequencingdemonstrated that GSE-BL intermediate was formed by self-cleavage and the maturation ofGSE-BL was stepwise. Mature GSE-BL could also be obtained without introducingexogenous trypsin when Lys at-1position in GSE-BL precursor was replaced by Glu, thusproviding a new strategy for the preparation of mature GSE-BL with enzymatic activity, theactivity of resultant mature GSE-BL towards Z-Phe-Leu-Glu-pNA also decreased by27.4%compared with native mature GSE-BL with His-tag, demonstrating that Lys-1Glu mutationhad slight negative effect on correct refolding of GSE-BL. Mature GSE-BL withoutpropeptide was majorly expressed as soluble form in E.coli, the purified product of whichshowed little activity towards Z-Phe-Leu-Glu-pNA;27kD intermediate was expressed asinclusion bodies, corresponding product after trypsin treatment had little activity towardsZ-Phe-Leu-Glu-pNA, demonstrating the presence of propeptide was indispensable for thecorrect refolding of GSE-BL to form active conformation.
The investigation on characteristic of GSE-BL implied us that GSE-BL had an optimalreaction pH of8.5, an optimal reaction temperature of37℃，and the Km of GSE-BL towardsZ-Phe-Leu-Glu-pNA was1.495±0.034mM, the Vmaxof which was50.237μmol/mg.minThe presence of2mM calcium ion could significantly enhance the thermostability ofGSE-BL at50℃, and EDTA could partially inhibite the activity of GSE-BL. The catalytic efficiency of GSE-BL towards Z-Phe-Leu-Glu-pNA was925fold as that of GSE-BL towardsZ-Asp-pNA.
A computer model of GSE-BL based on glutamate-specific endopeptidase from Bacillusintermidius (GSE-BI) was built. And we predicted that His47, Asp96and Ser167constitutestable catalytic triad of GSE-BL by the proton transport, site-directed mutagenesis wasperformed to prove this prediction. The results of site-directed mutagenesis also demonstratedthat Val2, Cys32, Arg89and His190was essential for the formation of catalytic triad andspecificity of GSE-BL. Val2was involved in the formation of substrate binding pocketing,and it is important for the correct folding of GSE-BL. Cys32binded withZ-Phe-Leu-Glu-pNA and was involved in the formation of disulfide bond. Arg89providedpositive charge for substrate binding pocket, facilitating the entrance of substrate; His190binded with the γ-carboxyl of Glu residue, compensating for the negative charge of Gluresidue. Cys181Ala mutation decreased the enzymatic activity and thermostability of GSE-BLtowards Z-Phe-Leu-Glu-pNA. The catalytic efficiency of GSE-BL towardsZ-Phe-Leu-Glu-pNA was enhanced50%by Phe57Ala mutation. The Asp51Arg, Glu101Alaand Glu104Arg mutation resulted in the destabilization of GSE-BL catalytic triad becauseof unbalanced charge distribution.
In this study, GSE-BL was applied to the removal of affinity tag of recombinant proteinand the maturation of bioactive peptide.
Vitreoscilla hemoglobin is a kind of hemoglobin that can enhance oxygen uptake rate.VHb-His6was expressed in E.coli as soluble form. His-Tag of VHb-His6could be removedwith a ratio of91.6%by GSE-BL treatment for15min, thus obtaining tagless VHb with thesame amino acid sequence as native VHb. The yield of tagless VHb was31.8mg/L. TaglessVHb showed higer affinity for carbonic oxide than VHb-His6. The Vmaxof tagless VHbtowards hydrogen peroxide was1169μmol/g.min, while the Vmaxof VHb-His6towardshydrogen peroxide was781μmol/g.min. And the welan gum yield could be increased16.2%by the addition of0.1mM tagless VHb to fermentation liquid of Alcaligenes ATCC31555,while the addition of0.1mM VHb-His6only increased welan gum by4.9%.
Human glucagon can stimulate the liver glycogenolysis, thus enhancing blood sugar levels.Human glucagon fused with N-His6tag was expressed in Pichia pastoris as secreted forms insupernatant. His-Tag of recombinant human glucagon could be removed with a ratio of91.6%by GSE-BL treatment for1h, and the molecular weight of tagless human glucagon was thesame as the standard synthesized human glucagon. The results of HPLC illustrated thatpurified human glucagon had the same retetion time with the standard synthesized human glucagon. The yield of tagless glucagons was50.5mg/L.
Human beta-defensin-2(HBD-2) is a kind of cysteine-rich and cationic antimicrobialpeptide, and Glu residue was introduced as recognition site before mature HBD-2, andHis-Tag was fused at N-terminus to facilitate purification. Mature HBD-2was obtain fromHBD-2precursor by GSE-BL treatment for1h, and the yield of which was23.2mg/L.Mature HBD-2exhibited higher antimicrobial activity towards Pseudomonas aeruginosa thanstaphylococcus aureas, and higer cytotoxity of mature HBD-2for HCT-8was observed thancytoxity of mature HBD-2for dendritic cells from mice marrow.
引文
[1] H. Neurath, Evolution of proteolytic enzymes. Science (New York, NY)224(1984)350-358.
[2] M.M. Krem, E. Di Cera, Molecular markers of serine protease evolution. The EMBO journal20(2001)3036-3045.
[3] L. Hedstrom, Serine protease mechanism and specificity. Chemical reviews102(2002)4501-4524.
[4] V. Fremeaux-Bacchi, M. Dragon-Durey, J. Blouin, C. Vigneau, D. Kuypers, B. Boudailliez, C. Loirat, E.Rondeau, W. Fridman, Complement factor I: a susceptibility gene for atypical haemolytic uraemic syndrome.Journal of medical genetics41(2004) e84-e84.
[5] L.R. Lund, K.A. Green, A.A. Stoop, M. Ploug, K. Almholt, J. Lilla, B.S. Nielsen, I.J. Christensen, C.S. Craik,Z. Werb, Plasminogen activation independent of uPA and tPA maintains wound healing in gene-deficient mice.The EMBO journal25(2006)2686-2697.
[6] D. Blow, P. Boyer, The Enzymes, Vol. Ill,3rd ed, PD Boyer, Ed, Academic, New York (1971)181-212.
[7] A. Warshel, G. Naray-Szabo, F. Sussman, J. Hwang, How do serine proteases really work? Biochemistry28(1989)3629-3637.
[8] H. Czapinska, J. Otlewski, Structural and energetic determinants of the S1‐site specificity in serineproteases. European Journal of Biochemistry260(2001)571-595.
[9] L. Gráf, á. Jancsó, L. Szilágyi, G. Hegyi, K. Pintér, G. Náray-Szabó, J. Hepp, K. Medzihradszky, W.J.Rutter, Electrostatic complementarity within the substrate-binding pocket of trypsin. Proceedings of the NationalAcademy of Sciences85(1988)4961-4965.
[10] A.J. Bridges, J.R. Knowles, An examination of the utility of photogenerated reagents by usingα-chymotrypsin. Biochemical Journal143(1974)663.
[11] L.T.J. Delbaere, W.L.B. Hutcheon, M.N.G. James, W.E. Thiessen, Tertiary structural differences betweenmicrobial serine proteases and pancreatic serine enzymes.(1975)758-763.
[12] G.R. Drapeau, Y. Boily, J. Houmard, Purification and properties of an extracellular protease ofStaphylococcus aureus. Journal of Biological Chemistry247(1972)6720-6726.
[13] J. Houmard, G.R. Drapeau, Staphylococcal protease: a proteolytic enzyme specific for glutamoyl bonds.Proceedings of the National Academy of Sciences69(1972)3506-3509.
[14] G. Drapeau, Role of metalloprotease in activation of the precursor of staphylococcal protease. Journal ofbacteriology136(1978)607-613.
[15] G.R. Drapeau, The primary structure of staphylococcal protease. Canadian journal of biochemistry56(1978)534-544.
[16] N. Khaidarova, G. Rudenskaya, L. Revina, V. Stepanov, N. Egorov, Glu, Asp-specific proteinase fromStreptomyces thermovulgaris. Biokhimiya54(1989)46-53.
[17] N. Yoshida, S. Tsuruyama, K. Nagata, K. Hirayama, K. Noda, S. Makisumi, Purification andcharacterization of an acidic amino acid specific endopeptidase of Streptomyces griseus obtained from acommercial preparation (Pronase). Journal of biochemistry104(1988)451-456.
[18] I. Svendsen, M.R. Jensen, K. Breddam, The primary structure of the glutamic acid-specific protease ofStreptomyces griseus. FEBS letters292(1991)165-167.
[19] S. Kakudo, N. Kikuchi, K. Kitadokoro, T. Fujiwara, E. Nakamura, H. Okamoto, M. Shin, M. Tamaki, H.Teraoka, H. Tsuzuki, Purification, characterization, cloning, and expression of a glutamic acid-specific proteasefrom Bacillus licheniformis ATCC14580. Journal of Biological Chemistry267(1992)23782-23788.
[20] K. Kitadokoro, E. Nakamura, M. Tamaki, T. Horii, H. Okamoto, M. Shin, T. Sato, T. Fujiwara, H. Tsuzuki,N. Yoshida, Purification, characterization and molecular cloning of an acidic amino acid-specific proteinasefrom Streptomyces fradiae ATCC14544. Biochimica et Biophysica Acta (BBA)-Protein Structure andMolecular Enzymology1163(1993)149-157.
[21] I. Leshchinskaya, E. Shakirov, E. Itskovitch, N. Balaban, A. Mardanova, M. Sharipova, M. Viryasov, G.Rudenskaya, V. Stepanov, Glutamyl endopeptidase of Bacillus intermedius, strain3-19. FEBS letters404(1997)241.
[22] I. Leshchinskaya, E. Shakirov, E. Itskovitch, N. Balaban, A. Mardanova, M. Sharipova, E. Blagova, V.Levdikov, I. Kuranova, G. Rudenskaya, Glutamyl endopeptidase of Bacillus intermedius strain3-19. Purification,properties, and crystallization. Biochemistry Biokhimiia62(1997)903.
[23] E. Itskovich, N. Balaban, A. Mardanova, E. Shakirov, M. Sharipova, I. Leshchinskaya, A. Ksenofontov, G.Rudenskaya, Enzymatic properties of thiol-dependent serine proteinase of Bacillus intermedius3-19.Biochemistry Biokhimiia62(1997)49.
[24] H. Okamoto, T. Fujiwara, E. Nakamura, T. Katoh, H. Iwamoto, H. Tsuzuki, Purification andcharacterization of a glutamic-acid-specific endopeptidase from Bacillus subtilis ATCC6051; application to therecovery of bioactive peptides from fusion proteins by sequence-specific digestion. Applied Microbiology andBiotechnology48(1997)27-33.
[25] K. Yokoi, M. Kakikawa, H. Kimoto, K. Watanabe, H. Yasukawa, A. Yamakawa, A. Taketo, K.I. Kodaira,Genetic and biochemical characterization of glutamyl endopeptidase of Staphylococcus warneri M. Gene281(2001)115-122.
[26] Y. Ohara-Nemoto, T. Ono, Y. Shimoyama, S. Kimura, T.K. Nemoto, Homologous and heterologousexpression and maturation processing of extracellular glutamyl endopeptidase of Staphylococcus epidermidis.Biological chemistry389(2008)1209-1217.
[27] N.A. Thornberry, H.G. Bull, J.R. Calaycay, K.T. Chapman, A.D. Howard, M.J. Kostura, D.K. Miller, S.M.Molineaux, J.R. Weidner, J. Aunins, A novel heterodimeric cysteine protease is required forinterleukin-1βprocessing in monocytes. Nature356(1992)768-774.
[28] N.A. Thornberry, T.A. Rano, E.P. Peterson, D.M. Rasper, T. Timkey, M. Garcia-Calvo, V.M. Houtzager,P.A. Nordstrom, S. Roy, J.P. Vaillancourt, A combinatorial approach defines specificities of members of thecaspase family and granzyme B Functional relationships established for key mediators of apoptosis. Journal ofBiological Chemistry272(1997)17907-17911.
[29] M.S. Lall, C. Karvellas, J.C. Vederas, β-Lactones as a new class of cysteine proteinase inhibitors: inhibitionof hepatitis A virus3C proteinase by N-Cbz-serine β-lactone. Organic letters1(1999)803-806.
[30] S.E. Webber, K. Okano, T.L. Little, S.H. Reich, Y. Xin, S.A. Fuhrman, D.A. Matthews, R.A. Love, T.F.Hendrickson, A.K. Patick, Tripeptide aldehyde inhibitors of human rhinovirus3C protease: design, synthesis,biological evaluation, and cocrystal structure solution of P1glutamine isosteric replacements. Journal ofmedicinal chemistry41(1998)2786-2805.
[31] I. Svendsen, K. Breddam, Isolation and amino acid sequence of a glutamic acid specific endopeptidasefrom Bacillus licheniformis. European Journal of Biochemistry204(2005)165-171.
[32] Y. Ohara-Nemoto, Y. Ikeda, M. Kobayashi, M. Sasaki, S. Tajika, S. Kimura, Characterization andmolecular cloning of a glutamyl endopeptidase from Staphylococcus epidermidis. Microbial pathogenesis33(2002)33-41.
[33] S. Fidalgo, F. Vázquez, M.C. Mendoza, F. Pérez, F.J. Méndez, Bacteremia Due to Staphylococcusepldermidis: Microbiologic, Epidemiologic, Clinical, and Prognostic Features. Review of Infectious Diseases12(1990)520-528.
[34] S. Takeda, G.B. Pier, Y. Kojima, M. Tojo, E. Muller, T. Tosteson, D.A. Goldmann, Protection againstendocarditis due to Staphylococcus epidermidis by immunization with capsular polysaccharide/adhesin.Circulation84(1991)2539-2546.
[35] M.E. Rupp, J.S. Ulphani, P.D. Fey, D. Mack, Characterization of Staphylococcus epidermidisPolysaccharide Intercellular Adhesin/Hemagglutinin in the Pathogenesis of Intravascular Catheter-AssociatedInfection in a Rat Model. Infection and immunity67(1999)2656-2659.
[36] D.V. Rebrikov, T.V. Akimkina, A.B. Shevelev, I.V. Demidyuk, A.M. Bushueva, S.V. Kostrov, G.G.Chestukhina, V.M. Stepanov, Bacillus intermedius glutamyl endopeptidase. Molecular cloning and nucleotidesequence of the structural gene. Journal of protein chemistry18(1999)21-27.
[37] J. Alexandra, R. Barbosa, J.W. Saldanha, R.C. Garratt, Novel features of serine protease active sites andspecificity pockets: sequence analysis and modelling studies of glutamate-specific endopeptidases andepidermolytic toxins. Protein engineering9(1996)591-601.
[38] V.L. Nienaber, K. Breddam, J.J. Birktoft, A glutamic acid specific serine protease utilizes a novel histidinetriad in substrate binding. Biochemistry32(1993)11469-11475.
[39] R. Meijers, E.V. Blagova, V.M. Levdikov, G.N. Rudenskaya, G.G. Chestukhina, T.V. Akimkina, S.V.Kostrov, V.S. Lamzin, I.P. Kuranova, The crystal structure of glutamyl endopeptidase from Bacillus intermediusreveals a structural link between zymogen activation and charge compensation. Biochemistry43(2004)2784-2791.
[40] J. Cavarelli, G. Prévost, W. Bourguet, L. Moulinier, B. Chevrier, B. Delagoutte, A. Bilwes, L. Mourey, S.Rifai, Y. Piémont, The structure of Staphylococcus aureus epidermolytic toxin A, an atypic serine protease, at1.7resolution. Structure5(1997)813-824.
[41] H.R. Stennicke, K. Breddam, J.J. Birktoft, Characterization of the S1binding site of the glutamicacid-specific protease from Streptomyces griseus. Protein science5(2008)2266-2275.
[42] J. Birktoft, D. Blow, Structure of crystalline-chymotrypsin. V. The atomic structure of tosyl-chymotrypsinat2resolution. Journal of molecular biology68(1972)187-240.
[43] L. Prasad, Y. Leduc, K. Hayakawa, L.T.J. Delbaere, The structure of a universally employed enzyme: V8protease from Staphylococcus aureus. Acta Crystallographica Section D: Biological Crystallography60(2004)256-259.
[44] I. Demidyuk, D. Romanova, E. Nosovskaya, G. Chestukhina, I. Kuranova, S. Kostrov, Modification ofsubstrate-binding site of glutamyl endopeptidase from Bacillus intermedius. Protein Engineering Design andSelection17(2004)411-416.
[45] M. Koch, C. Breithaupt, R. Kiefersauer, J. Freigang, R. Huber, A. Messerschmidt, Crystal structure ofprotoporphyrinogen IX oxidase: a key enzyme in haem and chlorophyll biosynthesis. The EMBO journal23(2004)1720-1728.
[46] F.X. Gomis-Rüth, M. Gómez, W. Bode, R. Huber, F. Avilés, The three-dimensional structure of the nativeternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C.The EMBO journal14(1995)4387.
[47] S. Kakudo, N. Kikuchi, K. Kitadokoro, T. Fujiwara, E. Nakamura, H. Okamoto, M. Shin, M. Tamaki, H.Teraoka, H. Tsuzuki, Purification, characterization, cloning, and expression of a glutamic acid-specific proteasefrom Bacillus licheniformis ATCC14580. Journal of Biological Chemistry267(1992)23782-23788.
[48] P. Nacharaju, A. Acharya, Hemoglobin semisynthesis. Critical Review in Biochemistry (1999)151-208.
[49] G. Sahni, Y.J. Cho, K.S. Iyer, S.A. Khan, R. Seetharam, A.S. Acharya, Semisynthetic hemoglobin A:reconstitution of functional tetramer from semisynthetic. alpha.-globin. Biochemistry28(1989)5456-5461.
[50] G. Sahni, A.K. Mallia, A.S. Acharya, Proteosynthetic activity of immobilized Staphylococcus aureus V8protease: Application in the semisynthesis of molecular variants of α-globin. Analytical biochemistry193(1991)178-185.
[51] M. Inouye, Intramolecular chaperone: the role of the pro-peptide in protein folding. Enzyme45(1991)314.
[52] Y. Ohta, H. Hojo, S. Aimoto, T. Kobayashi, X. Zhu, F. Jordan, M. Inouye, Pro-peptide as an intermolecularchaperone: renaturation of denatured subtilisin E with a synthetic pro-peptide. Molecular microbiology5(2006)1507-1510.
[53] I.V. Demidyuk, A.V. Shubin, E.V. Gasanov, S.V. Kostrov, Propeptides as modulators of functional activityof proteases. BioMol Concepts1(2010)305-322.
[54] K. Yoshikawa, H. Tsuzuki, T. Fujiwara, E. Nakamura, H. Iwamoto, K. Matsumoto, M. Shin, N. Yoshida, H.Teraoka, Purification, characterization and gene cloning of a novel glutamic acid-specific endopeptidase fromStaphylococcus aureus ATCC12600. Biochimica et Biophysica Acta (BBA)-Protein Structure and MolecularEnzymology1121(1992)221-228.
[55] C.H. Park, S.J. Lee, S.G. Lee, W.S. Lee, S.M. Byun, Hetero-and autoprocessing of the extracellularmetalloprotease (Mpr) in Bacillus subtilis. Journal of bacteriology186(2004)6457-6464.
[56] E. Gasanov, I. Demidyuk, A. Shubin, V. Kozlovskiy, O. Leonova, S. Kostrov, Hetero-and auto-activationof recombinant glutamyl endopeptidase from Bacillus intermedius. Protein Engineering Design and Selection21(2008)653-658.
[57] T.K. Nemoto, Y. Ohara‐Nemoto, T. Ono, T. Kobayakawa, Y. Shimoyama, S. Kimura, T. Takagi,Characterization of the glutamyl endopeptidase from Staphylococcus aureus expressed in Escherichia coli.FEBS Journal275(2008)573-587.
[58] J.J. Birktoft, K. Breddam,[8] Glutamyl endopeptidases. Methods in enzymology244(1994)114-126.
[59] S.B. S rensen, T.L. S rensen, K. Breddam, Fragmentation of proteins by S. aureus strain V8protease:Ammonium bicarbonate strongly inhibits the enzyme but does not improve the selectivity for glutamic acid.FEBS letters294(1991)195-197.
[60] W. Gebauer, J.R. Harris, G. Geisthardt, J. Markl, Keyhole Limpet Hemocyanin Type2(KLH2): Detectionand Immunolocalization of a Labile Functional Unit h. Journal of Structural Biology128(1999)280-286.
[61]M. Schuster, A. Aaviksaar, H.D. Jakubke, Enzyme-catalyzed peptide synthesis in ice. Tetrahedron46(1990)8093-8102.
[62] M. H nsler, H.D. Jakubke, Nonconventional protease catalysis in frozen aqueous solutions. Journal ofPeptide Science2(1996)279-289.
[63] S. Kumaran, D. Datta, R.P. Roy, Conformationally driven protease-catalyzed splicing of peptide segments:V8protease-mediated synthesis of fragments derived from thermolysin and ribonuclease A. Protein science6(2008)2233-2241.
[64] G. Ullmann, M. Haensler, W. Gruender, M. Wagner, H.J. Hofmann, H.D. Jakubke, Influence offreeze-concentration effect on proteinase-catalysed peptide synthesis in frozen aqueous systems. Biochimica etBiophysica Acta (BBA)-Protein Structure and Molecular Enzymology1338(1997)253-258.
[65] J.A. Zavadzkas, R.A. Plyler, S. Bouges, C.N. Koval, W.T. Rivers, C.U. Beck, E.I. Chang, R.E. Stroud, R.Mukherjee, F.G. Spinale, Cardiac-restricted overexpression of extracellular matrix metalloproteinase inducercauses myocardial remodeling and dysfunction in aging mice. American Journal of Physiology-Heart andCirculatory Physiology295(2008) H1394-H1402.
[66] D. Vercaigne‐Marko, E. Kosciarz, N. Nedjar-Arroume, D. Guillochon, Improvement of Staphylococcusaureus-V8-protease hydrolysis of bovine haemoglobin by its adsorption on to a solid phase in the presence ofSDS: peptide mapping and obtention of two haemopoietic peptides. Biotechnology and applied biochemistry31(2000)127-134.
[67] R. Froidevaux, F. Krier, N. Nedjar-Arroume, D. Vercaigne-Marko, E. Kosciarz, C. Ruckebusch, P.Dhulster, D. Guillochon, Antibacterial activity of a pepsin-derived bovine hemoglobin fragment. FEBS letters491(2001)159-163.
[68] P.L. Huang, Y. Sun, H.C. Chen, H.F. Kung, S. Lee-Huang, Proteolytic fragments of anti-HIV andanti-tumor proteins MAP30and GAP31are biologically active. Biochemical and biophysical researchcommunications262(1999)615-623.
[69] R. IPSEN, J. OTTE, K. B QVIST, Molecular self-assembly of partially hydrolysed α-lactalbumin resultingin strong gels with a novel microstructure. Journal of dairy research68(2001)277-286.
[70] J.F. Graveland-Bikker, R. Ipsen, J. Otte, C.G. de Kruif, Influence of calcium on the self-assembly ofpartially hydrolyzed α-lactalbumin. Langmuir20(2004)6841-6846.
[71] R. Ipsen, J. Otte, Self-assembly of partially hydrolysed α-lactalbumin. Biotechnology Advances25(2007)602-605.
[72] J. Otte, R. Ipsen, R. Bauer, M.J. Bjerrum, R. Waninge, Formation of amyloid-like fibrils upon limitedproteolysis of bovine α-lactalbumin. International dairy journal15(2005)219-229.
[73] J. Otte, R. Ipsen, A.M. Ladefoged, J. Srensen, Protease-induced aggregation of bovine-lactalbumin:Identification of the primary associating fragment. Journal of dairy research71(2004)88-96.
[74] R. Ipsen, J. Otte, Nano-structuring by means of proteolysis: rheology of novel gels from α-lactalbumin.Annu Trans Nordic Rheol Soc11(2003)89-94.
[75] L. Gul'ko, K. Voyushin, F. fluer, N. okorokova, M. krivenko, V. veiko, V. debabov, tumor-addressedgenetically engineered preparations used in cancer immunotherapy. ii. cloning of the pro-enterotoxin h (seh)gene from staphylococcus aureus and expression of this gene in escherichia coli. the study of enterotoxin hexcretion by E. coli cells. Biotechnology (2003)81-89.
[76] J. Ishizaki, M. Tamaki, M. Shin, H. Tsuzuki, K. Yoshikawa, H. Teraoka, N. Yoshida, Production ofrecombinant human glucagon in the form of a fusion protein in Escherichia coli; recovery of glucagon bysequence-specific digestion. Applied Microbiology and Biotechnology36(1992)483-486.
[77] F. Widmer, S. Bayne, G. Houen, B. Moss, R. Rigby, R. Whittaker, J. Johansen, Use of proteolytic enzymesfor the synthesis of fragments of mouse epidermal growth factor. Peptides (1984)193-200.
[78] M. Haensler, H.D. Wissmann, N. Wehofsky, Enzymatic formation of Glu-Xaa and Asp-Xaa bonds usingGlu/Asp‐specific endopeptidase from Bacillus licheniformis in frozen aqueous systems. Journal of PeptideScience6(2000)366-371.
[79] N. Wehofsky, N. Koglin, S. Thust, F. Bordusa, Reverse proteolysis promoted by in situ generated peptideester fragments. Journal of the American Chemical Society125(2003)6126-6133.
[80] R. Grünberg, I. Domgall, R. Günther, K. Rall, H.J. Hofmann, F. Bordusa, Peptide bond formation mediatedby substrate mimetics. European Journal of Biochemistry267(2000)7024-7030.
[81] V. eěovsky, H.D. Jakubke, Acyl transfer reactions catalyzed by native and modified α-chymotrypsin inacetonitrile with low water content. Enzyme and microbial technology16(1994)596-601.
[82] J. Bongers, W. Liu, T. Lambros, K. Breddam, R.M. Campbell, A.M. Felix, E.P. Heimer, Peptide synthesiscatalyzed by the Glu/Asp-specific endopeptidase. International Journal of Peptide and Protein Research44(1994)123-129.
[83] J. Bongers, T. Lambros, W. Liu, M. Ahmad, R.M. Campbell, A.M. Felix, E.P. Heimer, Enzymicsemisynthesis of a superpotent analog of human growth hormone-releasing factor. Journal of medicinalchemistry35(1992)3934-3941.
[84] S.S. Rajan, H. Lackland, S. Stein, D.T. Denhardt, Presence of an N-Terminal Polyhistidine Tag FacilitatesStable Expression of an Otherwise Unstable N-Terminal Domain of Mouse Tissue Inhibitor ofMetalloproteinase-1in Escherichia coli. Protein Expression and Purification13(1998)67-72.
[85] Q.M. Sun, L.L. Chen, L. Cao, L. Fang, C. Chen, Z.C. Hua, An Improved Strategy for High‐LevelProduction of Human Vasostatin120–180. Biotechnology progress21(2008)1048-1052.
[86] W. Tang, Z.Y. Sun, R. Pannell, V. Gurewich, J.N. Liu, An efficient system for production of recombinanturokinase-type plasminogen activator. Protein Expression and Purification11(1997)279-283.
[87] G. Kou, S. Shi, H. Wang, M. Tan, J. Xue, D. Zhang, S. Hou, W. Qian, S. Wang, J. Dai, Preparation andcharacterization of recombinant protein ScFv (CD11c)-TRP2for tumor therapy from inclusion bodies inEscherichia coli. Protein Expression and Purification52(2007)131-138.
[88] A. Mayer, S. Sharma, B. Tolner, N. Minton, D. Purdy, P. Amlot, G. Tharakan, R. Begent, K. Chester,Modifying an immunogenic epitope on a therapeutic protein: a step towards an improved system forantibody-directed enzyme prodrug therapy (ADEPT). British journal of cancer90(2004)2402-2410.
[89] H. Chen, Z. Xu, N. Xu, P. Cen, Efficient production of a soluble fusion protein containing humanbeta-defensin-2in E. coli cell-free system. Journal of biotechnology115(2005)307-315.
[90] D. Esposito, D.K. Chatterjee, Enhancement of soluble protein expression through the use of fusion tags.Current opinion in biotechnology17(2006)353-358.
[91] A. Chant, C.M. Kraemer-Pecore, R. Watkin, G.G. Kneale, Attachment of a histidine tag to the minimal zincfinger protein of the Aspergillus nidulans gene regulatory protein AreA causes a conformational change at theDNA-binding site. Protein Expression and Purification39(2005)152-159.
[92] A. Goel, D. Colcher, J.S. Koo, B.J.M. Booth, G. Pavlinkova, S.K. Batra, Relative position of thehexahistidine tag effects binding properties of a tumor-associated single-chain Fv construct. Biochimica etBiophysica Acta (BBA)-General Subjects1523(2000)13-20.
[93] K.M. Kim, E.C. Yi, D. Baker, K.Y.J. Zhang, Post-translational modification of the N-terminal His taginterferes with the crystallization of the wild-type and mutant SH3domains from chicken src tyrosine kinase.Acta Crystallographica Section D: Biological Crystallography57(2001)759-762.
[94] A.C. Freydank, W. Brandt, B. Dr ger, Protein structure modeling indicates hexahistidine‐tag interferencewith enzyme activity. Proteins: Structure, Function, and Bioinformatics72(2008)173-183.
[95] S. Cadel, C. Gouzy-Darmon, S. Petres, C. Piesse, V.L. Pham, M.C. Beinfeld, P. Cohen, T. Foulon,Expression and purification of rat recombinant aminopeptidase B secreted from baculovirus-infected insect cells.Protein Expression and Purification36(2004)19-30.
[96] I. Fonda, M. Kenig, V. Gaberc-Porekar, P. Pristovaek, V. Menart, Attachment of histidine tags torecombinant tumor necrosis factor-alpha drastically changes its properties. Scient World J2(2002)1312.
[97] E.G.E. de Vries, M.N. de Hooge, J.A. Gietema, S. de Jong, Correspondence re: CG Ferreira et al.,Apoptosis: Target of Cancer Therapy. Clin. Cancer Res.,8:2024–2034,2002. Clinical Cancer Research9(2003)912-912.
[98] Z.S. Derewenda, The use of recombinant methods and molecular engineering in protein crystallization.Methods34(2004)354-363.
[99] G. C aga, D.E. Bochkariov, G.G. Jokhadze, J. Hopp, P. Nelson, Natural poly-histidine affinity tag forpurification of recombinant proteins on cobalt (II)-carboxymethylaspartate crosslinked agarose. Journal ofChromatography A864(1999)247-256.
[100] A. Knappik, A. Pluckthun, An improved affinity tag based on the flag (r) peptide for the detection andpurification of recombinant antibody fragments. Biotechniques17(1994)754-761.
[101] A. Einhauer, A. Jungbauer, The FLAG peptide, a versatile fusion tag for the purification of recombinantproteins. Journal of biochemical and biophysical methods49(2001)455-465.
[102] B. Cass, P.L. Pham, A. Kamen, Y. Durocher, Purification of recombinant proteins from mammalian cellculture using a generic double-affinity chromatography scheme. Protein Expression and Purification40(2005)77-85.
[103] A. Skerra, T.G.M. Schmidt,[18] Use of the Strep-tag and streptavidin for detection and purification ofrecombinant proteins. Methods in enzymology326(2000)271-304.
[104] W.C. Lee, K.H. Lee, Applications of affinity chromatography in proteomics. Analytical biochemistry324(2004)1-10.
[105] K. Terpe, Overview of tag protein fusions: from molecular and biochemical fundamentals to commercialsystems. Applied Microbiology and Biotechnology60(2003)523-533.
[106] R.T. Raines, M. McCormick, T.R. Van Oosbree, R.C. Mierendorf,[23] The S· tag fusion system forprotein purification. Methods in enzymology326(2000)362-376.
[107] X.Q. Liu, Protein-splicing intein: Genetic mobility, origin, and evolution. Annual review of genetics34(2000)61-76.
[108] M. Kavoosi, J. Meijer, E. Kwan, A.L. Creagh, D.G. Kilburn, C.A. Haynes, Inexpensive one-steppurification of polypeptides expressed in Escherichia coli as fusions with the family9carbohydrate-bindingmodule of xylanase10A from T. maritima. Journal of Chromatography B807(2004)87-94.
[109] C. Scheich, V. Sievert, K. Büssow, An automated method for high-throughput protein purification appliedto a comparison of His-tag and GST-tag affinity chromatography. BMC biotechnology3(2003)12-18.
[110] D.R. Smyth, M.K. Mrozkiewicz, W.J. McGrath, P. Listwan, B. Kobe, Crystal structures of fusion proteinswith large-affinity tags. Protein science12(2009)1313-1322.
[111] M. Dyson, S.P. Shadbolt, K. Vincent, R. Perera, J. McCafferty, Production of soluble mammalian proteinsin Escherichia coli: identification of protein features that correlate with successful expression. BMCbiotechnology4(2004)32-38.
[112] J.D. Fox, K.M. Routzahn, M.H. Bucher, D.S. Waugh, Maltodextrin-binding proteins from diverse bacteriaand archaea are potent solubility enhancers. FEBS letters537(2003)53-57.
[113] R.J. Jenny, K.G. Mann, R.L. Lundblad, A critical review of the methods for cleavage of fusion proteinswith thrombin and factor Xa. Protein Expression and Purification31(2003)1-11.
[114] S.H. Shahravan, X. Qu, I.S. Chan, J.A. Shin, Enhancing the specificity of the enterokinase cleavagereaction to promote efficient cleavage of a fusion tag. Protein Expression and Purification59(2008)314-319.
[115] O.W. Liew, J.P. Ching Chong, T.G. Yandle, S.O. Brennan, Preparation of recombinant thioredoxin fusedN-terminal proCNP: analysis of enterokinase cleavage products reveals new enterokinase cleavage sites. ProteinExpression and Purification41(2005)332-340.
[116] M.T.W. Hearn, D. Acosta, Applications of novel affinity cassette methods: use of peptide fusion handlesfor the purification of recombinant proteins. Journal of Molecular Recognition14(2001)323-369.
[117] M.H. Hefti, C.J.G. Van Vugt-Van der Toorn, R. Dixon, J. Vervoort, A novel purification method forhistidine-tagged proteins containing a thrombin cleavage site. Analytical biochemistry295(2001)180-185.
[118] J.J. Lichty, J.L. Malecki, H.D. Agnew, D.J. Michelson-Horowitz, S. Tan, Comparison of affinity tags forprotein purification. Protein Expression and Purification41(2005)98-105.
[119] S.Y. Kwon, Y.J. Choi, T.H. Kang, K.H. Lee, S.S. Cha, G.H. Kim, H.S. Lee, K.T. Kim, K.J. Kim, Highlyefficient protein expression and purification using bacterial hemoglobin fusion vector. Plasmid53(2005)274-282.
[120] V. Rubio, Y. Shen, Y. Saijo, Y. Liu, G. Gusmaroli, S.P. Dinesh-Kumar, X.W. Deng, An alternativetandem affinity purification strategy applied to Arabidopsis protein complex isolation. The Plant Journal41(2005)767-778.
[121] H. Mao, A self-cleavable sortase fusion for one-step purification of free recombinant proteins. ProteinExpression and Purification37(2004)253-263.
[122] M. Gehrmann, B.T. Do, M. Wagner, K.A. Zettlitz, R.E. Kontermann, G. Foulds, A.G. Pockley, G.Multhoff, A novel expression and purification system for the production of enzymatic and biologically activehuman granzyme B. Journal of immunological methods371(2011)8-17.
[123] H.E. Humphries, M. Christodoulides, J.E. Heckels, Expression of the class1outer-membrane protein ofNeisseria meningitidis in Escherichia coli and purification using a self-cleavable affinity tag. Protein Expressionand Purification26(2002)243-248.
[124] N. Abdullah, H.A. Chase, Removal of poly-histidine fusion tags from recombinant proteins purified byexpanded bed adsorption. Biotechnology and bioengineering92(2005)501-513.
[125] S. Blumberg, D. Ben Meir, Method for producing human growth hormone, EP Patent0,489,711,2003.
[126] M. Kenig, S. Peternel, V. Gaberc-Porekar, V. Menart, Influence of the protein oligomericity on final yieldafter affinity tag removal in purification of recombinant proteins. Journal of chromatography A1101(2006)293-306.
[127] L. Trachuk, A. Shcheglov, E. Milgotina, G. Chestukhina, In vitro maturation pathway of a glutamylendopeptidase precursor from Bacillus licheniformis. Biochimie87(2005)529-537.
[128] T.K. Nemoto, T. Ono, Y. Shimoyama, S. Kimura, Y. Ohara-Nemoto, Determination of three amino acidscausing alteration of proteolytic activities of staphylococcal glutamyl endopeptidases. Biological chemistry390(2009)277-285.
[129] S.Y. Lee, B.C. Stark, D.A. Webster, Structure–function studies of the Vitreoscilla hemoglobin D-region.Biochemical and biophysical research communications316(2004)1101-1106.
[130] R. Kaur, R. Pathania, V. Sharma, S.C. Mande, K.L. Dikshit, Chimeric Vitreoscilla hemoglobin (VHb)carrying a flavoreductase domain relieves nitrosative stress in Escherichia coli: new insight into the functionalrole of VHb. Applied and environmental microbiology68(2002)152-160.
[131] K.L. Dikshit, Y. Orii, N. Navani, S. Patel, H.Y. Huang, B.C. Stark, D.A. Webster, Site-directedmutagenesis of bacterial hemoglobin: the role of glutamine (E7) in oxygen-binding in the distal heme pocket.Archives of biochemistry and biophysics349(1998)161-166.
[132] P.S. Tsai, V. Hatzimanikatis, J.E. Bailey, Effect of Vitreoscilla hemoglobin dosage on microaerobicEscherichia coli carbon and energy metabolism. Biotechnology and bioengineering49(1996)139-150.
[133] X. Wu, Y. Chen, Y. Li, O. Li, L. Zhu, C. Qian, X. Tao, Y. Teng, Constitutive expression of Vitreoscillahaemoglobin in Sphingomonas elodea to improve gellan gum production. Journal of applied microbiology110(2011)422-430.
[134] V.Y. Tabakov, L. Emelyanova, S. Antonova, T. Voeikova, Effect of the Bacterial Hemoglobin vhbGeneon the Efficiency of Intergeneric Conjugation Escherichia coli–Streptomyces and Biosynthesis of Antibiotics inStreptomyces. Russian Journal of Genetics37(2001)332-334.
[135] W. Minas, P. Brünker, P.T. Kallio, J.E. Bailey, Improved erythromycin production in a geneticallyengineered industrial strain of Saccharopolyspora erythraea. Biotechnology progress14(1998)561-566.
[136] L.J. Chien, H.T. Chen, P.F. Yang, C.K. Lee, Enhancement of cellulose pellicle production byconstitutively expressing Vitreoscilla hemoglobin in Acetobacter xylinum. Biotechnology progress22(2006)1598-1603.
[137] H. Yu, Y. Shi, Y. Zhang, S. Yang, Z. Shen, Effect of Vitreoscilla hemoglobin biosynthesis in Escherichiacoli on production of poly (β-hydroxybutyrate) and fermentative parameters. FEMS microbiology letters214(2002)223-227.
[138] H. Zhu, S. Sun, S. Zhang, Enhanced production of total flavones and exopolysaccharides via Vitreoscillahemoglobin biosynthesis in Phellinus igniarius. Bioresource technology102(2011)1747-1751.
[139] I. Dogan, K.R. Pagilla, D.A. Webster, B.C. Stark, Expression of Vitreoscilla hemoglobin in Gordoniaamarae enhances biosurfactant production. Journal of industrial microbiology&biotechnology33(2006)693-700.
[140] Y. Su, X. Li, Q. Liu, Z. Hou, X. Zhu, X. Guo, P. Ling, Improved poly-γ-glutamic acid production bychromosomal integration of the Vitreoscilla hemoglobin gene (vgb) in Bacillus subtilis. Bioresource technology101(2010)4733-4736.
[141] M. Xu, Z. Rao, H. Xu, C. Lan, W. Dou, X. Zhang, J. Jin, Z. Xu, Enhanced production of L-arginine byexpression of Vitreoscilla hemoglobin using a novel expression system in Corynebacterium crenatum. Appliedbiochemistry and biotechnology163(2011)707-719.
[142] D.H. Suthar, B.B. Chattoo, Expression of Vitreoscilla hemoglobin enhances growth and levels ofα-amylase in Schwanniomyces occidentalis. Applied Microbiology and Biotechnology72(2006)94-102.
[143] S. Aydin, D.A. Webster, B.C. Stark, Nitrite inhibition of Vitreoscilla hemoglobin (VHb) in recombinant E.coli: direct evidence that VHb enhances recombinant protein production. Biotechnology progress16(2000)917-921.
[144] P.T. Kallio, J.E. Bailey, Intracellular Expression of VitreoscillaHemoglobin (VHb) Enhances Total ProteinSecretion and Improves the Production of α-Amylase and Neutral Protease in Bacillus subtilis. Biotechnologyprogress12(2008)31-39.
[145] M. Urgun-Demirtas, B. Stark, K. Pagilla, Use of genetically engineered microorganisms (GEMs) for thebioremediation of contaminants. Critical reviews in biotechnology26(2006)145-164.
[146] H. Geckil, A. Arman, S. Gencer, B. Ates, H. Ramazan Yilmaz, Vitreoscilla hemoglobin rendersEnterobacter aerogenes highly susceptible to heavy metals. BioMetals17(2004)715-723.
[147] P. Adlercreutz, B. Mattiasson, Oxygen supply to immobilized cells. Applied Microbiology andBiotechnology16(1982)165-170.
[148] J. Creteur, W. Sibbald, J.L. Vincent, Hemoglobin solutions-Not just red blood cell substitutes. Critical caremedicine28(2000)3025-3034.
[149] R.G. Thurman, H.G. Ley, R. Scholz, Hepatic microsomal ethanol oxidation. European Journal ofBiochemistry25(2005)420-430.
[150] M.J.O. Wakelam, G.J. Murphy, V.J. Hruby, M.D. Houslay, Activation of two signal-transduction systemsin hepatocytes by glucagon. Nature323(1986)68-71.
[151] E. Samols, G. Marri, V. Marks, Interrelationship of glucagon, insulin and glucose: the insulinogenic effectof glucagon. Diabetes15(1966)855-866.
[152] J. Harder, J. Bartels, E. Christophers, J. Schroder, A peptide antibiotic from human skin. Nature387(1997)861-861.
[153] D.M. Hoover, K.R. Rajashankar, R. Blumenthal, A. Puri, J.J. Oppenheim, O. Chertov, J. Lubkowski, Thestructure of human β-defensin-2shows evidence of higher order oligomerization. Journal of BiologicalChemistry275(2000)32911-32918.
[154] J.M. Schr der, Epithelial peptide antibiotics. Biochemical pharmacology57(1999)121-134.
[155] J. Harder, U. Meyer-Hoffert, K. Wehkamp, L. Schwichtenberg, J.M. Schr der, Differential gene inductionof human β-defensins (hBD-1,-2,-3, and-4) in keratinocytes is inhibited by retinoic acid. Journal of investigativedermatology123(2004)522-529.
[156] A. Lichtenstein, T. Ganz, M.E. Selsted, R.I. Lehrer, In vitro tumor cell cytolysis mediated by peptidedefensins of human and rabbit granulocytes. Blood68(1986)1407-1410.
[157] S. Johnstone, K. Gelmon, L. Mayer, R. Hancock, M. Bally, In vitro characterization of the anticanceractivity of membrane-active cationic peptides. I. Peptide-mediated cytotoxicity and peptide-enhanced cytotoxicactivity of doxorubicin against wild-type and p-glycoprotein over-expressing tumor cell lines. Anti-cancer drugdesign15(2000)151-160.