단백질을 가지고 실험을 하는 것은 DNA work보다 훨씬 더 힘듭니다. 단백질을 순수 정제하는 것은 정말 노가다(?)에 가깝죠. 그래서 많은 이들이 어떻게든 정제를 쉽게 해보기 위해 만들어낸 것이 affinity tag 입니다. 가장 대표적인 것이 6xHis-tag이고 MBP (maltose binding protein), GST, CBP (chitin binding protein) 등등 여러가지가 있습니다. 하지만 이런 tag이나 커다란 단백질이 붙어있으면 여러가지로 방해가 되기 때문에 이 tag을 제거하는 방법들이 고안되었습니다. 아래는 tag 제거에 사용되는 단백질 분해효소들입니다.
제 개인적으로는 TEV protease가 제일 좋더군요. Thrombin이나 PreScission은 좀 문제가 있었고, 최근에 나온 HRV 3C나 TagZyme은 사용해 보지 못했습니다. 그런데 Invitrogen에서 파는 TEV는 생각보다 별로였습니다. TEV protease는 원래 stability가 매우 낮은데 invitrogen의 것도 그렇게 좋지 못한 것 같습니다. 그냥 이거 개발한 사람에게 클론을 보내달라고 하면 보내줍니다. ^^
(위 그림은 인비트로젠의 브로셔 사진)
하지만 아래의 효소들은 다 나름대로 특징이 있습니다. 예를 들어 TEV나 HRV 3C는 효소 자체에 His-tag이 붙어있어서 His-tag 제거에는 최고입니다. His-tag가 제거된 단백질과 분리가 자연스럽게 되니까요.
Qiagen의 신제품 TagZyme은 N-말단부터 2개의 펩타이드를 자릅니다. 그러니까 N-terminal His-tag 제거에만 사용가능하고 His-tag 이후 몇개의 아미노산이 더 잘려나갈 수도 있습니다.
아래는 각 효소들과 간단한 특징입니다. 링크를 따라가시면 각 회사의 카탈로그나 제품 정보를 보실 수 있습니다.
1) PreScission Protease (GE Health Care) PreScission Protease is a genetically engineered fusion protein consisting of human rhinovirus 3C protease and GST (1). This protease was specifically designed to facilitate removal of the protease by allowing simultaneous protease immobilization and cleavage of GST fusion proteins produced from the pGEX-6P vectors pGEX-6P-1, pGEX-6P-2, and pGEX-6P-3; see pGEX Vectors (GST Gene Fusion System). PreScission Protease specifically cleaves between the Gln and Gly residues of the recognition sequence of LeuGluValLeuPheGln/GlyPro (2).
2) TEV (Invitrogen) AcTEV™ Protease is an enhanced form of Tobacco Etch Virus (TEV) protease that is highly site-specific, highly active, and significantly more stable than native TEV protease, resulting in long-term activity. AcTEV™ Protease specifically recognizes a seven amino acid (Glu-Asn-Leu-Tyr-Phe-Gln-Gly, cleaving between Gln and Gly), making it useful for removing affinity tags from fusion proteins (1,2). For proper cleavage, the protein of interest is expressed with an AcTEV™ (TEV) Protease recognition sequence located between it and the affinity tag. Subsequent incubation with AcTEV™ Protease releases the protein of interest from the fusion tag. This is an effective way to remove solubility, secretion, detection, and purification tags from recombinant proteins. AcTEV™ Protease features
Enhanced enzyme stability for prolonged protease activity Activity over a broad temperature range (+4°C to 30°C) Activity over a broad pH range (pH 6.0 to 8.5) A six-histidine sequence to facilitate its removal from the digested protein sample Greater than 85% single-band purity with no non-specific protease contamination
Restriction Grade Factor Xa is a highly purified enzyme isolated from bovine plasma and activated with Russell’s viper venom. The Novagen preparation is purified to near homogeneity and shows no secondary cleavage from contaminating proteases. The preparation is also functionally tested for activity with fusion proteins.
Like enterokinase, Factor Xa cleaves at the C-terminal side of its recognition sequence (IleGluGlyArg↓) and can, therefore, be used for removing all vector-encoded sequences from appropriately designed constructs.
4) Thrombin (Novagen, Sigma, etc) Restriction Grade Thrombin is qualified to specifically cleave target proteins containing the recognition sequence LeuValProArg↓GlySer. The preparation is functionally tested for activity with fusion proteins and is free of detectable contaminating proteases. Thrombin is supplied with 10X Thrombin Cleavage Buffer and a Cleavage Control Protein.
5) SUMO Protease (Invitrogen) SUMO Protease, also known as Ulp, is a recombinant fragment of ULP1 (Ubl-specific protease 1) from Saccharomyces cerevisiae. It is highly specific for the SUMO protein fusion, recognizing the tertiary structure of SUMO rather than an amino acid sequence.
6) HRV 3C Protease (Novagen) Recombinant type 14 3C protease from human rhinovirus (HRV 3C) is the newest addition to the Novagen brand line of restriction grade proteases. The recombinant protease is a highly purified recombinant 6XHis-fusion protein, which recognizes the same cleavage site as the native enzyme: LeuGluValLeuPheGln/GlyPro. The small, 22 KDa size of the protease, optimal activity at 4° C, high specificity, and His•Tag® fusion make HRV 3C protease an ideal choice for rapid removal of fusion tags. The pET expression vectors pET-47b(+) through pET-50b(+) incorporate the HRV 3C protease cleavage site in combinations with His•Tag, S•Tag, thioredoxin (Trx•Tag™), glutathione-S-transferase (GST•Tag™), and NusA (Nus•Tag™) sequences. The combination of pET-47b(+) to pET-50b(+) vectors for expression, HRV 3C protease for fusion tag cleavage, and Ni-NTA His•Bind® metal affinity chromatography for protein purification as well as protease and fusion tag removal allows production of recombinant proteins free of vector-encoded sequences. Supplied as: 2000 units/ml in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.5 mM THP, and 50% glycerol.
7) TagZyme, DAPase (Qiagen) The TAGZyme System removes N-terminal His tags from recombinant proteins with high specificityand efficiency. DAPase Enzyme is used to sequentially cleave off dipeptides from the N-terminus of a purified, His-tagged protein (see figure "His-tag Removal Using TAGZyme Enzymes A", steps 1 and 2). Digestion is halted when the enzyme reaches a “stop point”, an amino acid motif that cannot serve as a substrate (see table "DAPase stop points").
8) Enterokinase (Novagen) Recombinant Enterokinase (rEK) is a highly purified preparation of the catalytic subunit of recombinant bovine enterokinase, which recognizes the identical cleavage site as the native enzyme (i.e., AspAspAspAspLys↓) and has similar enzymatic activity. rEK exhibits superior rates of cleavage of fusion proteins containing the recognition sequence when compared to the native enzyme (Collins-Racie 1995). Novagen rEK is purified to near homogeneity and, unlike some preparations of native bovine enterokinase, exhibits no secondary cleavage arising from contaminating proteases. The preparation is also functionally tested for activity with fusion proteins.
