Expression and purification of influenza virus polymerase PA and crystal structure of complex of polypeptides at amino terminal of PA, carboxyl terminal of PA and amino terminal of PB1
An influenza virus, polymerase technology, applied in the direction of virus/phage, virus, antiviral agent, etc., can solve problems such as loss of protease activity and impact of polymerase function.
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Embodiment 1
[0151] Methods for expressing influenza virus PA and PB1 polypeptides:
[0152] One embodiment of the present invention is to divide PA into two segments for expression, so as to express the first 256 amino acid residue fragments and 257-716 amino acid residue fragments of the amino terminal of PA respectively, and encode the two protein polypeptides The gene fragments were respectively cloned into E. coli expression vectors for protein expression in bacteria. The N-terminal peptide of PA was purified from bacteria expressing the N-terminal (1-256 amino acids) of PA alone, and the N-terminal peptide of PA was used for protein crystallization. The carboxy-terminal expression bacteria of PA were collected by centrifugation and used for co-purification with PB1 N-terminal polypeptide.
[0153] The polypeptide containing the first 25 or 48 amino acids of the amino terminal of PB1 (excluding the first methylthio amino acid) is expressed in bacteria in the form of GST fusion prot...
Embodiment 2
[0165] PA C / PB1 N Crystallization of short peptide complexes:
[0166] Concentrate the complex of PA and PB1 polypeptides expressed and purified by the above method to a concentration of about 5-30 mg / mL, and use crystallization reagents (Screen Kit I / II, Index, etc. kits from companies such as Hampton Research) by the gas-phase hanging drop method Screening conditions for crystal growth. After preliminary screening, the inventors obtained initial crystals under various conditions of crystallization reagents.
[0167] Through further optimization, crystals with good appearance were obtained in sodium acetate solutions containing about 1 M under the condition of using different pH buffers (pH 4-9). Larger triangular pyramid-shaped crystals were obtained in a buffer (pH 4-9) containing sodium acetate (from Sigma) at a concentration of 1-1.3M, with a resolution of about 4 angstroms.
[0168] For data collection by X-ray diffraction, the crystals required for the diffra...
Embodiment 3
[0171] PA C / PB1 N Crystal structure of the short peptide complex:
[0172] A set of parent data with a resolution of 2.9 Å was first collected for crystals of the N-terminal 25 peptide complex of PA-PB1 using a FR-E X-ray diffractometer (from Rigaku) at a wavelength of 1.5418 Å. Then, using the synchrotron radiation instrument located at APS in Chicago, USA (line station number: SBC 19ID; detection screen: ADSC Q315), at wavelengths of 0.9783 and 0.9785 angstroms, two sets of peak and edge with a resolution of about 3.3 angstroms were collected. Crystallographic data of atomic selenium derivatives. Using HKL2000 (Otwinowski 1997) to process the three sets of data received, it was found that their space groups are all P4(1)2(1)2. Utilize the multi-wavelength anomalous scattering method (Hendrickson 1991) to calculate the phase, and use SHELXD (Sheldrick 1998) to find the selenium atom in the processed sca file. The protein itself contains 14 methionines, and the inv...
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