Expression and purification of recombinant human alpha1-proteinase inhibitor and its single amino acid substituted variants in Escherichia coli for enhanced stability and biological activity.


Abstract

Human alpha(1)-proteinase inhibitor (alpha(1)-PI) is the most abundant protease inhibitor found in the blood and expression of biologically active recombinant alpha(1)-PI has great potential in therapeutic applications. We report here the expression of a synthetic alpha(1)-PI gene and its variants in Escherichia coli. Modified alpha(1)-PI gene and its single amino acid variants were cloned in pMAL-c2X vector, which allowed expression of recombinant protein(s) as a fusion of maltose-binding protein (MBP) with factor Xa protease recognition site between the fusion partners. The synthetic gene(s) were expressed in different E. coli strains and maximum expression of recombinant alpha(1)-PI and variants up to 24% of total soluble protein (TSP) was achieved with engineered strain carrying extra copies of tRNAs for rare codons. Recombinant alpha(1)-PI protein(s) were purified by amylose affinity chromatography with high homogeneity and overall yield of about 7-9 mg l(-1) of bacterial culture (approximately 5.2 g wet cell mass). E. coli expressed recombinant alpha(1)-PI showed specific anti-elastase activity and appeared as a single band of approximately 45.0 kDa on SDS-PAGE. Primary structure of purified protein and integrity of N-terminus has been verified by mass spectrometric analysis. Recombinant alpha(1)-PI expressed in E. coli was fully intact having molecular mass similar to native unglycosylated protein purified from human plasma. Increased thermostability and specific activities of purified alpha(1)-PI variant proteins confirmed the stabilizing effect of incorporated mutations. Our results demonstrate efficient expression and purification of stable and biologically active alpha(1)-PI and its variants in E. coli for further therapeutic applications.

Submission Details

ID: EKcRDp3X4

Submitter: Shu-Ching Ou

Submission Date: Nov. 29, 2018, 3:57 p.m.

Version: 1

Publication Details
Agarwal S;Jha S;Sanyal I;Amla DV,J Biotechnol (2010) Expression and purification of recombinant human alpha1-proteinase inhibitor and its single amino acid substituted variants in Escherichia coli for enhanced stability and biological activity. PMID:20346993
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
3NDD 2010-07-28 1.5 Cleaved antitrypsin with P10 Pro, and P9-P6 Asp
5NBU 2018-03-21 1.67 Crystal structure of native alpha-1-antitrypsin with seven stabilising mutations
5NBV 2018-03-21 1.73 Crystal structure of native alpha-1-antitrypsin with seven stabilising mutations
3NE4 2011-12-14 1.81 1.8 Angstrom structure of intact native wild-type alpha-1-antitrypsin
4PYW 2015-06-10 1.91 1.92 angstrom crystal structure of A1AT:TTAI ternary complex
2QUG 2008-08-12 2.0 Crystal structure of alpha-1-antitrypsin, crystal form A
1QLP 1999-09-27 2.0 2.0 ANGSTROM STRUCTURE OF INTACT ALPHA-1-ANTITRYPSIN: A CANONICAL TEMPLATE FOR ACTIVE SERPINS
1HP7 2001-03-14 2.1 A 2.1 ANGSTROM STRUCTURE OF AN UNCLEAVED ALPHA-1-ANTITRYPSIN SHOWS VARIABILITY OF THE REACTIVE CENTER AND OTHER LOOPS
1IZ2 2003-02-11 2.2 Interactions causing the kinetic trap in serpin protein folding
3DRM 2009-03-31 2.2 2.2 Angstrom Crystal Structure of Thr114Phe Alpha1-Antitrypsin
1OPH 2003-08-05 2.3 NON-COVALENT COMPLEX BETWEEN ALPHA-1-PI-PITTSBURGH AND S195A TRYPSIN
3CWL 2008-09-23 2.44 Crystal structure of alpha-1-antitrypsin, crystal form B
3CWM 2008-09-23 2.51 Crystal structure of alpha-1-antitrypsin complexed with citrate
1EZX 2000-10-25 2.6 CRYSTAL STRUCTURE OF A SERPIN:PROTEASE COMPLEX
1QMB 2000-02-06 2.6 Cleaved alpha-1-antitrypsin polymer
1OO8 2003-08-05 2.65 CRYSTAL STRUCTURE OF A1PI-PITTSBURGH IN THE NATIVE CONFORMATION
3NDF 2010-07-28 2.7 Cleaved antitrypsin with P8-P6 Asp
1ATU 1997-08-20 2.7 UNCLEAVED ALPHA-1-ANTITRYPSIN
1PSI 1996-12-07 2.92 Intact recombined alpha1-antitrypsin mutant PHE 51 to LEU
7API 1990-10-15 3.0 THE S VARIANT OF HUMAN ALPHA1-ANTITRYPSIN, STRUCTURE AND IMPLICATIONS FOR FUNCTION AND METABOLISM
9API 1990-10-15 3.0 THE S VARIANT OF HUMAN ALPHA1-ANTITRYPSIN, STRUCTURE AND IMPLICATIONS FOR FUNCTION AND METABOLISM
1D5S 2000-04-02 3.0 CRYSTAL STRUCTURE OF CLEAVED ANTITRYPSIN POLYMER
8API 1990-10-15 3.1 THE S VARIANT OF HUMAN ALPHA1-ANTITRYPSIN, STRUCTURE AND IMPLICATIONS FOR FUNCTION AND METABOLISM
3DRU 2009-03-31 3.2 Crystal Structure of Gly117Phe Alpha1-Antitrypsin
2D26 2005-11-29 3.3 Active site distortion is sufficient for proteinase inhibit second crystal structure of covalent serpin-proteinase complex
5IO1 2016-06-08 3.34 CRYSTAL STRUCTURE OF RECOMBINANT HUMAN Z ALPHA-1-ANTITRYPSIN
1KCT 1997-01-11 3.46 ALPHA1-ANTITRYPSIN
3T1P 2011-08-17 3.9 Crystal structure of an alpha-1-antitrypsin trimer

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
193.2 A,B Alpha-1-antitrypsin Q5RCW5 A1AT_PONAB
188.8 A,B Alpha-1-antitrypsin P01010 A1AT_PAPAN
200.0 A,B Alpha-1-antitrypsin P01009 A1AT_HUMAN
192.2 A,B Alpha-1-antitrypsin O00394 A1AT_CHLAE