A long-acting, highly potent interferon alpha-2 conjugate created using site-specific PEGylation

Recombinant interferon alpha-2 (IFN-alpha2) is used clinically to treat a variety of viral diseases and cancers. IFN-alpha2 has a short circulating half-life, which necessitates frequent administration to patients. Previous studies showed that it is possible to extend the circulating half-life of IFN-alpha2 by modifying lysine residues of the protein with amine-reactive poly(ethylene glycol) (PEG) reagents. However, amine-PEGylated IFN-alpha2 comprises a heterogeneous product mixture with low specific activity due to the large number and critical locations of lysine residues in IFN-alpha2. In an effort to overcome these problems we determined the feasibility of creating site-specific, mono-PEGylated IFN-alpha2 analogues by introducing a free (unpaired) cysteine residue into the protein, followed by modification of the added cysteine residue with a maleimide-PEG reagent. IFN-alpha2 cysteine analogues were expressed in Escherichia coli and purified, and their in vitro bioactivities were measured in the human Daudi cell line growth inhibition assay. Several cysteine analogues were identified that do not significantly affect in vitro biological activity of IFN-alpha2. Certain of the cysteine analogues, but not wild-type IFN-alpha2, reacted with maleimide-PEG to produce mono-PEGylated proteins. The PEG-Q5C analogue retained high in vitro bioactivity (within 3- to 4-fold of wild-type IFN-alpha2) even when modified with 20- and 40-kDa PEGs. Pharmacokinetic experiments indicated that the 20-kDa PEG-Q5C and 40-kDa PEG-Q5C proteins have 20-fold and 40-fold longer half-lives, respectively, than IFN-alpha2 following subcutaneous administration to rats. These studies demonstrate the feasibility of using site-specific PEGylation technology to create a long-acting, mono-PEGylated IFN-alpha2 protein with high specific activity.     

聚乙二醇定点修饰集成干扰素突变体Ⅱ

目的：用聚乙二醇（PEG）修饰集成干扰素突变体Ⅱ（IFN-Con-m2,IIFNm2）,通过纯化获得新型修饰分子并对该分子进行抗胰蛋白酶水解稳定性及初步药代动力学研究。 方法：将mPEG20000定点偶联到IIFNm2的第86位Cys残基上,修饰后的产物经CM层析后,以SDS-PAGE考察其纯度,用WISH-VSV系统进行生物活性测定;在0.1%胰蛋白酶条件下考察体外抗酶解稳定性;并以SD大鼠进行初步药代动力学研究,绘制血药浓度-时间曲线。采用3P87软件进行数据拟合,分析药物动力学参数。 结果：干扰素修饰率约为50%,且绝大多数以单修饰体（mono-PEG- IIFNm2）形式存在;提纯后mono-PEG-IIFNm2 的纯度大于98%,比活性约为修饰前IIFNm2的1%。抗胰蛋白酶水解试验表明：30min后,IIFNm2抗病毒活性残留为8%,mono-PEG-IIFNm2为41%。初步药代动力学研究显示：IIFNm的消除半衰期为(1.57±0.34)h,mono-PEG-IIFNm2为(18.0±4.0)h。 结论：成功地偶联了PEG和IIFNm2,建立了mono-PEG-IIFNm2的纯化工艺,PEG修饰能增加IIFNm2的体外抗胰蛋白酶水解稳定性,并显著延长体内半衰期。     

Site-specific PEGylation of bone morphogenetic protein-2 cysteine analogues

Three cysteine analogues of bone morphogenetic protein (BMP)-2, BMP2A2C, BMP2N56C, and BMP2E96C, were generated in order to enable the attachment of SH-reactive poly(ethylene glycol) (PEG) at specific sites. Three different approaches (Ap) were used for SH-specific PEGylation: (Ap1) reaction of glutathione activated proteins with thiol PEG; (Ap2) reaction of DTT reduced proteins with orthopyridyl disulfide PEG; (Ap3) reaction of DTT reduced proteins with maleimide PEG. Non-, mono-, and di-PEGylated BMP-2 analogues could be separated by RP-HPLC. Trypsin digestion of PEGylated proteins and Trypsin and GluC double-digestion of N-ethylmaleimide-labeled proteins confirmed that the modifications were site-specific. Surface plasmon resonance analysis of type I and type II receptor binding of the PEGylated BMP-2 analogues revealed that all three PEGylation approaches were equivalent. PEGylation at positions 2 and 96 caused a similar decrease in receptor affinity. PEGylation at position 56 resulted in a larger decrease in affinity for both types of receptors. Mono-PEGylated BMP-2 analogues exhibited intermediate affinities in comparison with unmodified and di-PEGylated proteins. However, the biological activity of the PEGylated BMP-2 analogues as measured in alkaline phosphatase assay was higher than BMP-2 wild-type for the PEGylated BMP2A2C, slightly reduced for the BMP2N56C, and strongly reduced for the BMP2E96C. These results taken together indicate that specific attachment of PEG at engineered sites of BMP-2 is possible and that the attachment site is critical for biological activity. Furthermore, the biological activity of PEGylated BMP-2 analogues in cell culture seems to be determined not only by receptor affinity, but also by other factors such as protein solubility and stability. It is also discussed that the attached PEG interferes with the binding of BMP-2 to modulator proteins, co-receptors, or heparinic sites of proteoglycans in the extracellular matrix.     

Enhanced circulating half-life and antitumor activity of a site-specific pegylated interferon-alpha protein therapeutic

Recombinant interferon alpha-2 (IFN-alpha2) has proven useful for treating a variety of human cancers and viral diseases. IFN-alpha2 has a short circulating half-life in vivo, which necessitates daily or thrice weekly administration to patients. It is possible to extend the circulating half-life of IFN-alpha2 by random modification of lysine residues in the protein with polyethylene glycol (PEG); however, such preparations have heterogeneous structures and low specific activities, and may not provide optimal therapeutic benefits to patients. A long-acting, site-specific, monoPEGylated IFN-alpha2 protein has now been created by targeted attachment of a 20 kDa or a 40 kDa maleimide-PEG to a cysteine analogue of IFN-alpha2, M111C. In vitro bioactivities of the purified 20 kDa and 40 kDa PEG-M111C proteins were within 2- to 3-fold of those of wild type IFN-alpha2 and 7- to 10-fold better than that of a 40 kDa PEG IFN-alpha2 protein created using nontargeted, amine-PEGylation methodology. The 20 kDa and 40 kDa PEG-M111C proteins demonstrated 26- to 38-fold longer half-lives, respectively, than IFN-alpha2 following subcutaneous administration to rats. The 20 kDa PEG M111C protein inhibited growth of human NIH:OVCAR-3 cells transplanted into nude mice by >90%, as measured by tumor size, tumor weight, and number of animals with detectable tumors at necropsy, and was significantly more effective than a comparable dose of IFN-alpha2. These data extend our previous findings that bioactivity of IFN-alpha2 can be largely preserved by targeted attachment of PEG moieties to nonessential sites in the protein and provide evidence that site-specific PEGylated IFN-alpha2 proteins possess enhanced tumoricidal properties in vivo.     

Site-directed PEGylation of human basic fibroblast growth factor

Through site-directed mutagenesis, three cysteines of human basic fibroblast growth factor (hbFGF) were replaced with serine residues, resulting in a hbFGF mutant named hbFGFSer25,69,92. The mutant with only one cysteine residue at the 87th position, whose mitogenic activity was comparable to that of wild-type hbFGF, was further coupled to polyethylene glycol with a molecular size of 5 kDa (PEG5K) via the cysteine residue to obtain another hbFGF derivative, PEG5K-hbFGFSer25,69,92. The optimal modification reaction was conducted at 4 degrees C for 4 h at a molar ratio of PEG5K to hbFGFSer25,69,92 of 20:1. The result of SDS-PAGE showed that the modification extent was up to 80%. The modified product was purified by ion exchange chromatography. Compared to the hbFGF mutant, the purified PEG5K-hbFGFSer25,69,92 still retained about 60% of the mitogenic activity of the former, which provided a good basis for further studying the bioactivity of the PEGylated protein in vivo.     

Solid-phase N-terminus PEGylation of recombinant human fibroblast growth factor 2 on heparin-sepharose column

Recombinant fibroblast growth factor-2 (FGF-2) has been extensively studied and used in several clinical applications including wound healing, bone regeneration, and neuroprotection. Poly(ethylene glycol) (PEG) modification of recombinant human FGF-2 (rhFGF-2) in solution phase has been studied to increase the in vivo biostabilities and therapeutic potency. However, the solution-phase strategy is not site-controlled and the products are often not homogeneous due to the generation of multi-PEGylated proteins. In order to increase mono-PEGylated rhFGF-2 level, a novel solid-phase strategy for rhFGF-2 PEGylation is developed. RhFGF-2 proteins were loaded onto a heparin-sepharose column and the PEGylaton reaction was carried out at the N-terminus by PEG20 kDa butyraldehyde through reductive alkylation. The PEGylated rhFGF-2 was purified to near homogeneity by SP sepharose anion-exchange chromatography and the purity was more than 95% with a yield of mono-PEGylated rhFGF-2 of 58.3%, as confirmed by N-terminal sequencing and MALDI-TOF mass spectrometry. In vitro biophysical and biochemical measurements demonstrated that PEGylated rhFGF-2 has an unchanged secondary structure, receptor binding activity, cell proliferation, and MAP kinase stimulating activity, and an improved bio- and thermal stability. Animal assay showed that PEGylated rhFGF-2 has an increased half-life and reduced immunogenicity. Compared to conventional solution-phase PEGylation, the solid-phase PEGylation is advantageous in reaction time, production of mono-PEGylated protein, and improvement of biochemical and biological activity.     

溶葡球菌酶的定点突变与PEG巯基定点修饰

为了建立聚乙二醇 (PEG) 巯基定点修饰溶葡球菌酶的方法,并检验假定连接区的突变与修饰对酶活的影响,对溶葡球菌酶的假定连接区进行了巯基聚乙二醇定点修饰研究。通过分析溶葡球菌酶的结构特征,选择两个结构域之间的氨基酸 (133-154aa) 进行定点突变引入半胱氨酸残基。使用单甲氧基聚乙二醇马来酰亚胺 (mPEG-MAL) 进行定点修饰,对修饰后的酶进行纯化并测定酶活性。结果表明定点突变的半胱氨酸残基PEG修饰效率高、产物单一,运用简便的Ni2+-NTA柱亲和层析法实现了一步分离,获得了高纯度的目标蛋白,但在连接区进行定点突变及PEG定点修饰后的酶活有不同程度的降低,表明假定连接区部分位点的PEG修饰会对溶葡球菌酶的催化活性产生一定影响。     

Tailoring structure-function and pharmacokinetic properties of single-chain Fv proteins by site-specific PEGylation

The utility of single-chain Fv proteins as therapeutic agents would be realized if the circulating lives of these minimal antigen-binding polypeptides could be both prolonged and adjustable. We have developed a general strategy for creating tailored monoPEGylated single-chain antibodies. Free cysteine residues were engineered in an anti-TNF-alpha scFv at the C-terminus or within the linker segments of both scFv orientations, V(L)-linker-V(H) and V(H)-linker-V(L). High-level expression of 10 designed variant scFv proteins in Pichia pastoris allowed rapid purification. Optimization of site-specific conjugate preparation with 5, 20 and 40 kDa maleimide-PEG polymers was achieved and a comparison of the structural and functional properties of the scFv proteins and their PEGylated counterparts was performed. Peptide mapping and MALDI-TOF mass spectrometric analysis confirmed the unique attachment site for each PEG polymer. Independent biochemical and bioactivity analyses, including binding affinities and kinetics, antigenicity, flow cytometric profiling and cell cytotoxicity rescue, demonstrated that the functional activities of the 10 designed scFv conjugates are maintained, while scFv activity variations between these alternative assays can be correlated with conjugate and analytical designs. Pharmacokinetic studies of the PEGylated scFv in mice demonstrated up to 100-fold prolongation of circulating lives, in a range comparable to clinical antibodies.     

PEGylated murine Granulocyte-macrophage colony-stimulating factor: production, purification, and characterization

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates proliferation, differentiation, and function of hematopoietic progenitor cells. Aside from expansion of hematopoietic cells, GM-CSF has shown efficacy in other diseases, including Crohn's disease. While GM-CSF being clinically used in humans, the ability to perform mechanistic studies in murine models is difficult due to the limited availability and rapid clearance of murine GM-CSF in the peripheral blood. To address these issues, we efficiently expressed murine GM-CSF under the control of the AOX1 gene promoter in Pichia pastoris using the Mut(S) strain KM71H. We describe the unique conditions that are required for efficient production by high-density fermentation and purification of mGM-CSF protein. Recombinant mGM-CSF protein was purified by tangential flow ultrafiltration and preparative reverse phase chromatography. To address limited half life or rapid clearance in mice, recombinant murine GM-CSF was modified by lysine-directed polyethylene glycol conjugation (PEGylation). PEG-modified and unmodified proteins were characterized by amino terminus sequence analysis and matrix assisted laser desorption ionization time-of-flight mass spectrometry. Under the mild reaction conditions, the recombinant protein is efficiently modified by PEGylation on an average of 2-3 sites per molecule. In vivo treatment of mice with PEGylated mGM-CSF, but not the unmodified recombinant mGM-CSF, reproduces the potent colony stimulating effects of human GM-CSF in patients on myeloid progenitor populations, as assessed by FACs analysis. This simplified approach for the expression, purification, and modification of a biologically potent form of murine GM-CSF should facilitate the study of central mechanisms of action in murine disease models.     

集成干扰素突变体IIFN72C的构建及其聚乙二醇定点修饰

目的：设计构建集成干扰素突变体IIFN72C并进行聚乙二醇定点修饰,以获得高活性的长效干扰素分子。方法：利用蛋白质分子同源模建,选择在集成干扰素分子IIFN的第72位引入半胱氨酸残基构成集成干扰素突变体IIFN72C。诱导表达后经包涵体变复性和层析纯化,与单甲氧基聚乙二醇（mPEGMAL）定点偶联。修饰产物经纯化后,以SDSPAGE考察其纯度,用WISHVSV系统进行生物活性测定。结果：IIFN72C以包涵体形式表达,表达量占菌体总蛋白的30%以上,比活性与突变前相当;修饰产物大多数为单修饰体,纯化后纯度大于98%,比活性保留约为修饰前的8%。结论：成功设计并表达IIFN72C用于PEG定点修饰,修饰产物活性保留得以提高。     

Preparation, characterization and in vitro bioactivity of N-terminally PEGylated staphylokinase dimers

PEGylation can improve the therapeutic efficacy of proteins by increasing serum half-life of proteins and reducing immunogenicity and antigenicity. However, PEGylation results in a substantial loss of the bioactivity of proteins due to the steric hindrance of polyethylene glycol (PEG). Dimerization of the proteins is an efficient approach to increase the bioactivity of the PEG-protein conjugates. Here, staphylokinase (SAK) was used due to its therapeutic potential for coronary thrombolysis. SAK dimers (dSAK) were prepared by engineering cysteine residue at the C-terminus of SAK and dimerization of the cysteine residue with 1,4-bismaleimidobutane. PEG aldehyde was used for site-specific PEGylation of dSAK at one of its two N-termini. Structural analysis indicated that dimerization of SAK can decrease the steric hindrance of PEG and increase the binding affinity of PEG-SAK to plasminogen. Dimerization of SAK increased the relative bioactivity of PEG-SAK from 39.0% to 62.0%. Therefore, site-specifically PEGylated dSAK at one of its two N-termini has higher bioactivity than the N-terminal PEGylated SAK.     

Site-specific incorporation of PEGylated amino acids into proteins using nonnatural amino acid mutagenesis

Site-directed incorporation of PEGylated nonnatural amino acids with 4, 8, and 12 repeated ethylene glycol units was examined in a cell-free translation system. PEGylated aminophenylalanine derivatives were successfully incorporated into proteins, whereas PEGylated lysines were not. The incorporation efficiency of the PEGylated amino acids decreased with an increase in PEG chain length. The present method will be useful for preparation of proteins which are PEGylated in a site-specific and quantitative manner.     

蛋白药物的聚乙二醇定点修饰策略与最佳位点

聚乙二醇修饰是一种改善蛋白质药物临床药效行之有效的方法。聚乙二醇修饰具有延长蛋白质药物在体内的半衰期、降低免疫原性和延缓蛋白酶降解、提高稳定性和溶解性等优点。而聚乙二醇的定点修饰由于能够获得均一性和高活性保留率的产物,并能提高产率,已经引起了广泛关注。综述了近年来聚乙二醇定点修饰蛋白质药物方面的研究进展,着重介绍了聚乙二醇定点修饰的策略及最佳修饰位点,并对聚乙二醇定点修饰技术的发展趋势进行了展望。     

A Sequence-Specific RNA-Binding Protein Complements Apobec-1 To Edit Apolipoprotein B mRNA

The editing of apolipoprotein B (apo-B) mRNA involves the site-specific deamination of cytidine to uracil. The specificity of editing is conferred by an 11-nucleotide mooring sequence located downstream from the editing site. Apobec-1, the catalytic subunit of the editing enzyme, requires additional proteins to edit apo-B mRNA in vitro, but the function of these additional factors, known as complementing activity, is not known. Using RNA affinity chromatography, we show that the complementing activity binds to a 280-nucleotide apo-B RNA in the absence of apobec-1. The activity did not bind to the antisense strand or to an RNA with three mutations in the mooring sequence. The eluate from the wild-type RNA column contained a 65-kDa protein that UV cross-linked to apo-B mRNA but not to the triple-mutant RNA. This protein was not detected in the eluates from the mutant or the antisense RNA columns. Introduction of the mooring sequence into luciferase RNA induced cross-linking of the 65-kDa protein. A 65-kDa protein that interacted with apobec-1 was also detected by far-Western analysis in the eluate from the wild-type RNA column but not from the mutant RNA column. For purification, proteins were precleared on the mutant RNA column prior to chromatography on the wild-type RNA column. Silver staining of the affinity-purified fraction detected a single prominent protein of 65 kDa. Our results suggest that the complementing activity may function as the RNA-binding subunit of the holoenzyme.     

PEG chain length impacts yield of solid-phase protein PEGylation and efficiency of PEGylated protein separation by ion-exchange chromatography: Insights of mechanistic models

The mechanisms behind protein PEGylation are complex and dictated by the structure of the protein reactant. Hence, it is difficult to design a reaction process which can produce the desired PEGylated form at high yield. Likewise, efficient purification processes following protein PEGylation must be constructed on an ad hoc basis for each product. The retention and binding mechanisms driving electrostatic interaction-based chromatography (ion-exchange chromatography) of PEGylated proteins (randomly PEGylated lysozyme and mono-PEGylated bovine serum albumin) were investigated, based on our previously developed model Chem. Eng. Technol. 2005, 28, 1387–1393. PEGylation of each protein resulted in a shift to a smaller elution volume compared to the unmodified molecule, but did not affect the number of binding sites appreciably. The shift of the retention volume of PEGylated proteins correlated with the calculated thickness of PEG layer around the protein molecule. Random PEGylation was carried out on a column (solid-phase PEGylation) and the PEGylated proteins were separated on the same column. Solid-phase PEGylation inhibited the production of multi-PEGylated forms and resulted in a relatively low yield of selective mono-PEGylated form. Pore diffusion may play an important role in solid-phase PEGylation. These results suggest the possibility of a reaction and purification process development based on the mechanistic model for PEGylated proteins on ion exchange chromatography.     

Characterization of site-specific ScFv PEGylation for tumor-targeting pharmaceuticals

New radiopharmaceuticals are possible using site-specific conjugation of small tumor binding proteins and poly(ethylene glycol) (PEG) scaffolds to provide modular multivalent, homo- or heterofunctional cancer-targeting molecules having preferred molecular size, valence, and functionality. Residence time in plasma can be optimized by modification of the size, number, and charge of the protein units. However, random PEG conjugation (PEGylation) of these small molecules via amine groups has led to variations of structural conformation and binding affinity. To optimize PEGylation, scFvs have been recombinantly produced in a vector that adds an unpaired cysteine (c) near the scFv carboxy terminus (scFv-c), thus providing a specific site for thiol conjugation. To evaluate the general applicability of this unpaired cysteine for PEGylation of scFv-c, conjugation efficiency was determined for four different scFvs and several PEG molecules having thiol reactive groups. The effect of the PEG molecular format on scFv-c PEG malignant cell binding was also addressed. ScFvs produced as scFv-c and purified by anti E-TAG affinity chromatography were conjugated using PEG molecules with maleimide (Mal) or o-pyridyl disulfide (OPSS). Conjugations were performed at pH 7.0, with 2 molar excess TCEP/scFv and PEG-(Mal) or PEG-OPSS, using 5:1 (PEG/scFv). PEG-Mal conjugation efficiency was also evaluated with 1:5 (PEG/scFv). PEGylation efficiency was determined for each reaction by quantitation of the products on SDS-PAGE. ScFv-c conjugation with unifunctional maleimide PEGs resulted in PEG conjugates incorporating 30-80% of the scFv-c, but usually above 50%. Efficiency of scFv-c conjugation to both functional groups of the bifunctional PEG-(Mal)2 varied between the PEG and scFv-c molecules studied. A maximum of 45% of scFv-c protein was conjugated as PEG- (scFv-c)2 using the smallest PEG-(Mal)2 (2 kDa). No significant increase in scFv-c conjugation was observed by the use of greater than a 5 molar excess of PEG/scFv-c. Under the same conjugation conditions, PEG as OPSS yielded less than 10% PEG-scFv-c. PEG-(scFv)2 conjugates had increased binding in ELISA using malignant cell membranes, when compared with unmodified scFv-c. PEGylated-scFv binding was comparable with unmodified scFv-c. In summary, scFv-c can be PEGylated in a site-specific manner using uni- or bivalent PEG-Mal, either linear or branched. ScFv-c was most efficiently conjugated to smaller PEG-Mal molecules, with the smallest, 2 kDa PEG-Mal, usually PEGylating 60-90% of the scFv-c. ScFv-c conjugation to form PEG-(scFv-c)2 reached greatest efficiency at 45%, and its purified form demonstrated greater binding than the corresponding scFv-c.     

Reproducible preparation and effective separation of PEGylated recombinant human granulocyte colony-stimulating factor with novel "PEG-pellet" PEGylation mode and ion-exchange chromatography

A novel preparation for polyethylene glycol (PEG) derivatives and chromatographic separation procedure of the PEGylated recombinant human granulocyte colony-stimulating factor (rhG-CSF) were designed to evaluate the reproducibility and scalability at large laboratory-scale level. The new "PEG-pellet" PEGylation mode was successfully applied to control the pH fluctuation during the conjugation reaction, a general problem in traditional liquid-phase conjugation mode. Moreover, two consecutive ion-exchange chromatography steps were successfully used to separate and purify the PEGylated rhG-CSF. Cation-exchange chromatography was firstly applied to separate PEGylated rhG-CSF from intact rhG-CSF, followed by anion-exchange chromatography to obtain individual PEG-rhG-CSF species (mono-, di- and tri-PEGylated rhG-CSF) and remove the excess free PEG. Furthermore, the molecular weight of individual PEGylated rhG-CSF was identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and SDS-PAGE, and cell proliferation activity in vitro was detected by MTT assay using NFS-60 cell.     

Site-specific PEGylation of native disulfide bonds in therapeutic proteins

Native disulfide bonds in therapeutic proteins are crucial for tertiary structure and biological activity and are therefore considered unsuitable for chemical modification. We show that native disulfides in human interferon alpha-2b and in a fragment of an antibody to CD4(+) can be modified by site-specific bisalkylation of the two cysteine sulfur atoms to form a three-carbon PEGylated bridge. The yield of PEGylated protein is high, and tertiary structure and biological activity are retained.     

重组溶葡萄球菌酶的PEG定点修饰

为获得高抗菌活性聚乙二醇(PEG)定点修饰的溶葡萄球菌酶(Lysn),根据该酶的高级结构,在它的催化域和结合域上优选8个位点(Q9、N13、N40、T172、N174、G197、V240和T244),将其分别突变成半胱氨酸,纯化后的溶葡萄球菌酶突变体经DTT处理后与20 kDa的单甲氧基聚乙二醇马来酰亚胺(m PEG-MAL)进行定点修饰反应,RP-HPLC分析显示PEG修饰率大于70%,修饰产物经MacroCap SP阳离子交换层析纯化后,PEG化溶葡萄球菌酶的纯度大于95%。比浊法和最小抑菌浓度(MIC)实验表明20K-PEG-Lysn V240C和20K-PEG-Lysn T244C的抗菌活性维持在原来的50%左右。研究结果为溶葡萄球菌酶全身给药治疗金黄色葡萄球菌感染奠定基础。     

Autoprocessing of the HIV-1 protease using purified wild-type and mutated fusion proteins expressed at high levels in Escherichia coli

Various constructs of the human immunodeficiency virus, type 1 (HIV-1) protease containing flanking Pol region sequences were expressed as fusion proteins with the maltose-binding protein of the malE gene of Escherichia coli. The full-length fusion proteins did not exhibit self-processing in E. coli, thereby allowing rapid purification by affinity chromatography on cross-linked amylose columns. Denaturation of the fusion protein in 5 M urea, followed by renaturation, resulted in efficient site-specific autoprocessing to release the 11-kDa protease. Rapid purification involving two column steps gave an HIV-1 protease preparations of greater than 95% purity (specific activity approximately 8500 pmol.min-1.micrograms protease-1) with an overall yield of about 1 mg/l culture. Incubation of an inactive mutant protease fusion protein with the purified wild-type protease resulted in specific trans cleavage and release of the mutant protease. Analysis of products of the HIV-1 fusion proteins containing mutations at either the N- or the C-terminal protease cleavage sites indicated that blocking one of the cleavage sites influences the cleavage at the non-mutated site. Such mutated full-length and truncated protease fusion proteins possess very low levels of proteolytic activity (approximately 5 pmol.min-1.micrograms protein-1).